Unidirectional Reconstitution into Detergent-destabilized Liposomes of the Purified Lactose Transport System of Streptococcus thermophilus

The lactose transport protein (LacS) of Streptococcus thermophilus was amplified to levels as high as 8 and 30% of total membrane protein in Escherichia coli and S. thermophilus, respectively. In both organisms the protein was functional and the expression levels were highest with the streptococcal lacS promoter. Also a LacS deletion mutant, lacking the carboxyl-terminal regulatory domain, could be amplified to levels >20% of membrane protein. Membranes from S. thermophilus proved to be superior in terms of efficient solubilization and ease and extent of purification of LacS; >95% of LacS was solubilized with relatively low concentrations of Triton X-100, n-octyl-β-D-glucoside, n-dodecyl-β-D-maltoside, or C12E8. The LacS protein carrying a poly-histidine tag was purified in large quantities (~5 mg/liter of culture) and with a purity >98% in a two-step process involving nickel chelate affinity and anion exchange chromatography. The membrane reconstitution of LacS was studied systematically by stepwise solubilization of preformed liposomes, prepared from E. coli phospholipid and phosphatidylcholine, and protein incorporation at the different stages of liposome solubilization. The detergents were removed by adsorption onto polystyrene beads and H+-lactose symport and lactose counterflow were measured. Highest transport activities were obtained when Triton X-100 was used throughout the solubilization/purification procedure, whereas activity was lost irreversibly with n-octyl-β-D-glucoside. For reconstitutions mediated by n-dodecyl-β-D-maltoside, C12E8, and to a lesser extent Triton X-100, the highest transport activities were obtained when the liposomes were titrated with low amounts of detergent (onset of liposome solubilization). Importantly, under these conditions proteoliposomes were obtained in which LacS was reconstituted in an inside-out orientation, as suggested by the outside labeling of a single cysteine mutant with a membrane impermeable biotin-maleimide. The results are consistent with a mechanism of reconstitution in which the hydrophilic regions of LacS prevent a random insertion of the protein into the membrane. Consistent with the in vivo lactose/galactose exchange catalyzed by the LacS protein, the maximal rate of lactose counterflow was almost 2 orders of magnitude higher than that of H+-lactose symport.

Black carbon as an additional indicator of the adverse health effects of airborne particles compared with PM10 and PM2.5.

Current air quality standards for particulate matter (PM) use the PM mass concentration [PM with aerodynamic diameters ≤ 10 μm (PM(10)) or ≤ 2.5 μm (PM(2.5))] as a metric. It has been suggested that particles from combustion sources are more relevant to human health than are particles from other sources, but the impact of policies directed at reducing PM from combustion processes is usually relatively small when effects are estimated for a reduction in the total mass concentration

Establishing the phenotypic spectrum of ZTTK syndrome by analysis of 52 individuals with variants in SON

Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome, an intellectual disability syndrome first described in 2016, is caused by heterozygous loss-of-function variants in SON. Its encoded protein promotes pre-mRNA splicing of many genes essential for development. Whereas individual phenotypic traits have previously been linked to erroneous splicing of SON target genes, the phenotypic spectrum and the pathogenicity of missense variants have not been further evaluated. We present the phenotypic abnormalities in 52 individuals, including 17 individuals who have not been reported before. In total, loss-of-function variants were detected in 49 individuals (de novo in 47, inheritance unknown in 2), and in 3, a missense variant was observed (2 de novo, 1 inheritance unknown). Phenotypic abnormalities, systematically collected and analyzed in Human Phenotype Ontology, were found in all organ systems. Significant inter-individual phenotypic variability was observed, even in individuals with the same recurrent variant (n = 13). SON haploinsufficiency was previously shown to lead to downregulation of downstream genes, contributing to specific phenotypic features. Similar functional analysis for one missense variant, however, suggests a different mechanism than for heterozygous loss-of-function. Although small in numbers and while pathogenicity of these variants is not certain, these data allow for speculation whether de novo missense variants cause ZTTK syndrome via another mechanism, or a separate overlapping syndrome. In conclusion, heterozygous loss-of-function variants in SON define a recognizable syndrome, ZTTK, associated with a broad, severe phenotypic spectrum, characterized by a large inter-individual variability. These observations provide essential information for affected individuals, parents, and healthcare professionals to ensure appropriate clinical management

Mapping carcass and meat quality QTL on Sus Scrofa chromosome 2 in commercial finishing pigs

Quantitative trait loci (QTL) affecting carcass and meat quality located on SSC2 were identified using variance component methods. A large number of traits involved in meat and carcass quality was detected in a commercial crossbred population: 1855 pigs sired by 17 boars from a synthetic line, which where homozygous (A/A) for IGF2. Using combined linkage and linkage disequilibrium mapping (LDLA), several QTL significantly affecting loin muscle mass, ham weight and ham muscles (outer ham and knuckle ham) and meat quality traits, such as Minolta-L* and -b*, ultimate pH and Japanese colour score were detected. These results agreed well with previous QTL-studies involving SSC2. Since our study is carried out on crossbreds, different QTL may be segregating in the parental lines. To address this question, we compared models with a single QTL-variance component with models allowing for separate sire and dam QTL-variance components. The same QTL were identified using a single QTL variance component model compared to a model allowing for separate variances with minor differences with respect to QTL location. However, the variance component method made it possible to detect QTL segregating in the paternal line (e.g. HAMB), the maternal lines (e.g. Ham) or in both (e.g. pHu). Combining association and linkage information among haplotypes improved slightly the significance of the QTL compared to an analysis using linkage information only

Detergent-Mediated Reconstitution of Membrane Proteins

The efficiency of reconstitution of the lactose transport protein (LacS) of Streptococcus thermophilus is markedly higher with Triton X-100 than with other detergents commonly employed to mediate the membrane insertion. To rationalize these differences, the lipid/detergent structures that are formed in the reconstitution process were studied by cryotransmission electron microscopy. Surprisingly, the two nonionic detergents Triton X-100 and n-dodecyl β-D-maltoside (DDM) affected the liposome structures in a completely different manner. Preformed liposomes titrated with Triton X-100 maintained their bilayer structure far beyond the onset of solubilization, and transport activity was maximal when LacS was inserted into these structures. With DDM the vesicular structures were already disrupted at the onset of solubilization and these membrane sheets were converted into long threadlike micelles at higher DDM to lipid ratios. Triton X-100 allowed the protein to be reconstituted with the hydrophilic surface exposed to the outside, whereas LacS was incorporated randomly when DDM was used. These differences in orientation are readily explained by the different lipid-detergent structures formed by Triton X-100 and DDM. The orientation of the reconstituted LacS protein is a critical factor for the activity of the protein as the kinetics of translocation is very different for opposite directions of transport.

Kinetic Analysis of Lactose and Proton Coupling in Glu379 Mutants of the Lactose Transport Protein of Streptococcus thermophilus

The role of Glu379 in the lactose-H+ symport protein (LacS) of Streptococcus thermophilus was studied by analyzing the kinetic mechanism of transport of wild-type and Ala379, Asp379, and Gln379 mutant proteins. Glu379 forms part of the sequence motif Lys-X-X-His-X-X-Glu that is present in a number of sugar transport proteins, including LacY of Escherichia coli. The E379A and E379Q mutants were defective in the uptake of lactose against a concentration gradient and lactose-dependent proton uptake, but catalyzed facilitated influx of lactose down a concentration gradient and equilibrium exchange with rates similar to that of the wild-type enzyme. The E379D mutant was partially defective in the coupled transport of lactose and protons. These results suggest that an acidic residue at position 379 is required for the coupled uptake of lactose and protons and are consistent with a mechanism in which lactose transport in the E379A and E379Q mutants occurs by uniport rather than proton symport. Lactose efflux down a concentration gradient in wild-type LacS and LacS-E379D increased with pH with apparent pK (pKa) values of ≥8.5 and 8.0, respectively, whereas efflux in the E379Q mutant increased sigmoidally with a pKa of about 6.0. Imposition of an artificial membrane potential (inside negative) in membrane vesicles bearing wild-type LacS or LacS-E379Q not only inhibited the lactose efflux mediated by wild-type but also that of the mutant enzyme. To associate the role of Glu379 with specific step(s) in the translocation cycle of LacS, the properties of wild-type LacS and the Glu379 mutants have been evaluated by numerical analysis of simple kinetic schemes for translocation catalysis by solute H+ symport proteins. The properties of the wild-type enzyme are consistent with a mechanism in which the order of ligand binding on the inside is substrate first and proton last, whereas the order is random (or proton first, substrate last) at the outer surface of the membrane. The wild-type enzyme is asymmetric with regard to proton binding; the pK for proton binding on the outside is at least 4 units higher than the pK on the inside. The properties of the Glu379 mutants correspond with a lowering of the pK on the outside (pKOUT ~ pKIN), and the induction of a leak pathway in which the binary enzyme-substrate complex becomes mobile.

The primary structures of pancreatic ribonucleases from African porcupine and casiragua, two hystricomorph rodent species

The amino acid sequences of the pancreatic ribonucleases from African porcupine (Hystrix cristata) and casiragua (Proechimys guairae, a caviomorph rodent species related to the coypu) were determined. The ribonucleases were isolated form minces of pancreatic tissue which had been used for the extraction of the insulins. The results of the sequence determinations of residues 67–78 in both enzymes were ambiguous. Therefore, homology with other ribonucleases has been used in deriving these sequences. At position 94 aspartic acid was found, while ali other ribonuclease sequenced to date have asparagine at this position. This may indicate a specific deamidation as a result of the acidic conditions during the extraction of insulin. The amino acid sequence of African porcupine ribonuclease shows a close relationship with those of the South-American caviomorph rodents, which implies that the hystricomorph suborder of the rodents, to which both the African porcupine and the caviomorphs belong, is a natural (evolutionary) taxon. Both porcupine and casiragua ribonuclease are glycoproteins with complex-type carbohydrate chains attached to asparagine34