The presenilin proteins are components of multiple membrane-bound complexes that have different biological activities

Several lines of evidence have indicated that the presenilin proteins function within macromolecular complexes and are necessary for the regulated intramembranous proteolysis of certain type 1 transmembrane proteins, including the amyloid precursor protein, Notch, and p75. Data from multiple complementary experiments now suggest that there may be several distinct presenilin complexes. We show here that presenilin mutations and certain detergents affect the abundance and componentry of the presenilin complexes, and these structural effects correlate with their effects on gamma-secretase activity. Our data suggest that there are at least three complexes, including a approximately 150-kDa nicastrin-aph-1 complex (which is likely to be a precursor complex). There is a stable and abundant intermediate complex of approximately 440 kDa, which contains aph-1, pen-2, nicastrin, and PS1. However, it is the very low abundance, high mass (>/=670 kDa) heteromeric complexes that are associated with the highest gamma-secretase-specific activity.     

Epoxidation of C-branched glycals: unexpected stereochemical results and their theoretical rationale

This paper describes the synthesis of C-3 methyl-branched glycosides by epoxidation of partially unblocked L-configured glycals. The stereochemical result depends on the orientation of the allylic hydroxyl group. A theoretical explanation is presented, based on the conformational preferences of the respective glycal half-chair conformations that were estimated by applying the BP density functional and a valence triple-zeta basis set.     

Glycosylphosphatidyl-inositols in murine malaria: Plasmodium yoelii yoelii

Glycosylphosphatidyl-inositols (GPIs) are vital major glycoconjugates in intraerythrocytic stages of Plasmodium. Here, we report on the biosynthesis and the characterization of GPIs synthesized by the murine malarial parasite P. yoelii yoelii YM. Parasitized erythrocytes were labeled in vivo and in vitro with either radioactive nucleotide sugar precursors, ethanolamine or glucosamine. The pathway leading to the formation of GPI precursors was found to resemble that described for P. falciparum; however, in P. yoelii, the formation of an additional hydrophilic precursor containing an acid-labile modification was detected. The data suggest that this modification is linked to the fourth mannose attached to the trimannosyl backbone in an alpha1-2 linkage. The modification was susceptible to hydrofluoric acid (HF), but not to nitrous acid (HNO(2)). Data obtained from size-exclusion chromatography on Bio-Gel P4, and Mono Q analysis of the fragments generated by HNO(2) deamination suggest that the modification is due to the presence of an additional ethanolamine linked to the fourth mannose via a phosphodiester bond.     

Environmental influences on signal transduction through membranes: a retrospective mini-review

This mini-review is addressed to the question how the membranous environment may affect traffic of receptors and signalling from membrane-anchored receptors on the outside of cells to transducers and targets on the inside. Signal transduction by membrane-anchored receptors to the interior of the cell and eventually to the genome is a central issue in cellular regulation. In this context the role of membrane fluidity and of the cytoskeleton in restricting the mobility of proteins are discussed and the evidence for a structural order in membranes which could limit the mobility of proteins is scrutinised.     

Mutational analysis of the human FATE gene in 144 infertile men

The FATE gene maps to Xq28 where one case of a translocation breakpoint has been found in an infertile man. Moreover, the FATE promoter contains a putative SF-1-binding site, and FATE has been proposed as representing a target gene of SF-1 in testicular development or germ cell differentiation. This study presents a complete mutational screening of the FATE gene in a random group of 144 infertile males. Four polymorphisms and two mutations were found. Three of the polymorphisms, viz., 741CT, 905AC, and 3985CT, occurred in exon 5 and intron 2 and did not alter the deduced polypeptide. One polymorphism resulted in the conservative amino acid exchange, A10 V, in 16.0% of the patients. This substitution occurred with similar frequencies in the control groups, indicating that the mutation does not affect fertility in men or women. The two mutations caused the non-conservative amino acid substitutions S125R (patient 1) and I34T (patient 2). A family study (patient 1) revealed, however, that S125R was inherited and that a fertile male family member carried the mutation. Patient 2 did not have relevant family members who could be examined. Thus, this study has shown that only 1.4% of infertile men have mutations in the FATE gene, and that some of these mutations do not singly cause infertility. Hence, FATE may not play an important role in the disease-state of infertile men attending fertility clinics. However, FATE mutations cannot be excluded as being a contributing factor in some cases of male infertility.     

Ontogenesis of epithelial phosphate transport systems in goats

The rapid development of precocial goats in the first weeks after birth requires an adequate adaptation of phosphate transport systems to maintain the P homeostasis at each developmental stage. Here we examined the age-related development of Na+-Pi transport systems in small intestines, kidneys, and parotid glands of goats. Kinetic parameters were determined by brush-border membrane vesicle uptake studies, and relative expression of NaPi type II mRNA and protein was recorded by molecular biological methods. High intestinal Pi transport capacity was already present on the first day of life. Within the first 3 wk of life there seemed to be a change in the type of Na+-dependent Pi transporter, and NaPi IIb was expressed increasingly up to the fifth month of life. Renal Na+-Pi transport capacity was also high at birth, and this was associated with high expression levels of NaPi IIa mRNA, indicating the important role of this transporter for renal Pi reabsorption. At weaning an increase in both intestinal and renal Na+-Pi transport balanced the increasing requirements for Pi to establish the endogenous Pi cycle. Salivary Pi concentration and parotid NaPi II mRNA rose markedly to guarantee an adequate Pi supply for rumen microbes. We concluded that the high demand for Pi in young goats was assured by high basal Na+-Pi transport capacity of small intestines and kidney expressed continuously during ontogenesis.     

Functional analysis of BamHI DNA cytosine-N4 methyltransferase

We show that the kinetic mechanism of the DNA (cytosine-N(4)-)-methyltransferase M.BamHI, which modifies the underlined cytosine (GGATCC), differs from cytosine C(5) methyltransferases, and is similar to that observed with adenine N(6) methyltransferases. This suggests that the obligate order of ternary complex assembly and disassembly depends on the type of methylation reaction. In contrast, the single-turnover rate of catalysis for M.BamHI (0.10s(-1)) is closer to the DNA (cytosine-C(5)-)-methyltransferases (0.14s(-1)) than the DNA (adenine-N(6)-)-methyltransferases (>200s(-1)). The nucleotide flipping transition dominates the single-turnover constant for adenine N(6) methyltransferases, and, since the disruption of the guanine-cytosine base-pair is essential for both types of cytosine DNA methyltransferases, this transition may be a common, rate-limiting step for methylation for these two enzyme subclasses. The similar overall rate of catalysis by M.BamHI and other DNA methyltransferases is consistent with a common rate-limiting catalytic step of product dissociation. Our analyses of M.BamHI provide functional insights into the relationship between the three different classes of DNA methyltransferases that complement both prior structural and evolutionary insights.     

Probing the sensory rhodopsin II binding domain of its cognate transducer by calorimetry and electrophysiology

Sensory rhodopsin II, a repellent phototaxis receptor from Natronobacterium pharaonis (NpSRII) forms a tight complex with its cognate transducer (NpHtrII). Light excitation of the receptor triggers conformational changes in both proteins, thereby activating the cellular two-component signalling cascade. In membranes, the two proteins form a 2:2 complex, which dissociates to a 1:1 heterodimer in micelles. Complexed to the transducer sensory rhodopsin II is no longer capable of light-driven proton pumping. In order to elucidate the dimerisation and the size of the receptor-binding domain of the transducer, isothermal titration calorimetry and electrophysiological experiments have been carried out. It is shown, that an N-terminal sequence of 114 amino acid residues is sufficient for tight binding (K(d)=240nM; DeltaH=-17.6kJmol(-1)) and for inhibiting the proton transfer. These data and results obtained from selected site-directed mutants indicate a synergistic interplay of transducer transmembrane domain (1-82) and cytoplasmic peptide (83-114) leading to an optimal and specific interaction between receptor and transducer.     

Different heparan sulfate proteoglycans serve as cellular receptors for human papillomaviruses

Papillomaviruses replicate in stratified epithelia of skin and mucosa. Infection with certain human papillomavirus (HPV) types is the main cause of anogenital neoplasia, in particular cervical cancer. Early events of papillomavirus infectivity are poorly understood. While heparan sulfate proteoglycans (HSPGs) mediate initial binding to the cell surface, the class of proteins carrying heparan sulfates has not been defined. Here we examined two processes of papillomavirus infection, attachment of virus-like particles (VLP) to cells and infection with authentic HPV type 11 (HPV11) virions. Of the HSPGs, syndecan-1 is the major epithelial form and is strongly upregulated in wound edge keratinocytes. We employed K562 cells, which lack HSPGs except minor amounts of endogenous betaglycan, and stable clones that express cDNAs of syndecan-1, syndecan-4, or glypican-1. Binding of VLP correlated with levels of heparan sulfate on the cell surface. Parental K562 bound HPV16 VLP weakly, whereas all three K562 transfectants demonstrated enhanced binding, with the highest binding capacity observed for syndecan-1-transfected cells, which also expressed the most HSPG. For HPV11 infectivity assays, a high virion inoculum was required to infect K562 cells, whereas ectopic expression of syndecan-1 increased permissiveness eightfold and expression of syndecan-4 or glypican-1 fourfold. Infection of keratinocytes was eliminated by treatment with heparitinase, but not phospholipase C, further implicating the syndecan family of integral membrane proteins as receptor proteins. Human keratinocytes with a homozygous deletion of alpha6 integrin are permissive for HPV11 infection. These results indicate that several HSPGs can serve as HPV receptors and support a putative role for syndecan-1, rather than alpha6 integrin, as a primary receptor protein in natural HPV infection of keratinocytes.     

Control of water uptake by rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.): role of the outer part of the root

A new pressure-perfusion technique was used to measure hydraulic and osmotic properties of the outer part of roots (OPR) of 30-day-old rice plants (lowland cultivar: IR64, and upland cultivar: Azucena). The OPR comprised rhizodermis, exodermis, sclerenchyma and one cortical cell layer. The technique involved perfusion of aerenchyma of segments from two different root zones (20-50 mm and 50-100 mm from the tip) at precise rates using aerated nutrient solution. The hydraulic conductivity of the OPR (Lp(OPR)=1.2x10(-6) m s(-1) MPa(-1)) was larger by a factor of 30 than the overall hydraulic conductivity (Lp(r)=4x10(-8) m s(-1) MPa(-1)) as measured by pressure chamber and root pressure probe. Low reflection coefficients were obtained for mannitol and NaCl for the OPR (sigma(sOPR)=0.14 and 0.09, respectively). The diffusional water permeability ( P(dOPR)) estimated from isobaric flow of heavy water was smaller by three orders of magnitude than the hydraulic conductivity (Lp(OPR)/ P(fOPR)). Although detailed root anatomy showed well-defined Casparian bands and suberin lamellae in the exodermis, the findings strongly indicate a predominantly apoplastic water flow in the OPR. The Lp(OPR) of heat-killed root segments increased by a factor of only 2, which is in line with the conclusion of a dominating apoplastic water flow. The hydraulic resistance of the OPR was not limiting the passage of water across the root cylinder. Estimations of the hydraulic properties of aerenchyma suggested that the endodermis was rate-limiting the water flow, although the aerenchyma may contribute to the overall resistance. The resistance of the aerenchyma was relatively low, because mono-layered cortical septa crossing the aerenchyma ('spokes') short-circuited the air space between the stele and the OPR. Spokes form hydraulic bridges that act like wicks. Low diffusional water permeabilities of the OPR suggest that radial oxygen losses from aerenchyma to medium are also low. It is concluded that in rice roots, water uptake and oxygen retention are optimized in such a way that hydraulic water flow can be kept high in the presence of a low efflux of oxygen which is diffusional in nature.