In vitro synthesis of lactose permease to probe the mechanism of membrane insertion and folding

Insertion and folding of polytopic membrane proteins is an important unsolved biological problem. To study this issue, lactose permease, a membrane transport protein from Escherichia coli, is transcribed, translated, and inserted into inside-out membrane vesicles in vitro. The protein is in a native conformation as judged by sensitivity to protease, binding of a monoclonal antibody directed against a conformational epitope, and importantly, by functional assays. By exploiting this system it is possible to express the N-terminal six helices of the permease (N(6)) and probe changes in conformation during insertion into the membrane. Specifically, when N(6) remains attached to the ribosome it is readily extracted from the membrane with urea, whereas after release from the ribosome or translation of additional helices, those polypeptides are not urea extractable. Furthermore, the accessibility of an engineered Factor Xa site to Xa protease is reduced significantly when N(6) is released from the ribosome or more helices are translated. Finally, spontaneous disulfide formation between Cys residues at positions 126 (Helix IV) and 144 (Helix V) is observed when N(6) is released from the ribosome and inserted into the membrane. Moreover, in contrast to full-length permease, N(6) is degraded by FtsH protease in vivo, and N(6) with a single Cys residue at position 148 does not react with N-ethylmaleimide. Taken together, the findings indicate that N(6) remains in a hydrophilic environment until it is released from the ribosome or additional helices are translated and continues to fold into a quasi-native conformation after insertion into the bilayer. Furthermore, there is synergism between N(6) and the C-terminal half of permease during assembly, as opposed to assembly of the two halves as independent domains.     

Identification of a novel system L amino acid transporter structurally distinct from heterodimeric amino acid transporters

A cDNA that encodes a novel Na+-independent neutral amino acid transporter was isolated from FLC4 human hepatocarcinoma cells by expression cloning. When expressed in Xenopus oocytes, the encoded protein designated LAT3 (L-type amino acid transporter 3) transported neutral amino acids such as l-leucine, l-isoleucine, l-valine, and l-phenylalanine. The LAT3-mediated transport was Na+-independent and inhibited by 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, consistent with the properties of system L. Distinct from already known system L transporters LAT1 and LAT2, which form heterodimeric complex with 4F2 heavy chain, LAT3 was functional by itself in Xenopus oocytes. The deduced amino acid sequence of LAT3 was identical to the gene product of POV1 reported as a prostate cancer-up-regulated gene whose function was not determined, whereas it did not exhibit significant similarity to already identified transporters. The Eadie-Hofstee plots of LAT3-mediated transport were curvilinear, whereas the low affinity component is predominant at physiological plasma amino acid concentration. In addition to amino acid substrates, LAT3 recognized amino acid alcohols. The transport of l-leucine was electroneutral and mediated by a facilitated diffusion. In contrast, l-leucinol, l-valinol, and l-phenylalaninol, which have a net positive charge induced inward currents under voltage clamp, suggesting these compounds are transported by LAT3. LAT3-mediated transport was inhibited by the pretreatment with N-ethylmaleimide, consistent with the property of system L2 originally characterized in hepatocyte primary culture. Based on the substrate selectivity, affinity, and N-ethylmaleimide sensitivity, LAT3 is proposed to be a transporter subserving system L2. LAT3 should denote a new family of organic solute transporters.     

Effects of leptin on hypothalamic arcuate neurons in Wistar and Zucker rats: an in vitro study

Leptin, a product of the ob gene, decreases food intake and body weight in both Wistar and Zucker obese rats when administered centrally or peripherally. To examine whether these leptin effects might be mediated through a neuropeptide Y (NPY) signaling pathway in the medial part of the arcuate nucleus of the hypothalamus (vmARC), the effects of leptin on vmARC neurons in Wistar and Zucker obese rats were examined electrophysiologically using brain slice preparations. Bath application of leptin inhibited about 60% of the vmARC neurons recorded in slices from Wistar rats. Similar inhibitory effects of leptin on vmARC neurons were also observed under low-Ca2+, high-Mg2+ Ringer's solution. However, inhibitory effects were almost absent under Ringer's solution containing a protein kinase C inhibitor, chelerythrine chloride. In slices from Zucker obese rats, leptin inhibited only about 25% of the vmARC neurons recorded, and the proportion of neurons inhibited was significantly smaller for these rats than for Wistar rats. These results suggest that reductions in food intake and body weight induced by leptin in both Wistar and Zucker obese rats are partly mediated via inhibition of an NPY signaling pathway in the vmARC.     

Histopathological studies of senile plaques and cerebral amyloidosis in cynomolgus monkeys

Senile plaques (SPs) and cerebral amyloid angiopathy (CAA), pathological hallmarks of Alzheimer's disease, have not been thoroughly investigated histopathologically in nonhuman primates. To determine the onset age and histopathological characteristics of SPs and CAA, we examined the brains of 64 cynomolgus monkeys (Macaca fascicularis) from 2 to 35 years old. Mature (classical and primitive) plaques appeared in 16 out of 25 monkeys that were >20 years old. Moreover, mature plaques were observed more frequently than diffuse plaques and were located in the temporal cortex of the superior or inferior gyri and amygdala. Diffuse plaques in contrast to mature plaques did not show definite tendencies in onset age and distribution. CAA appeared in more than 22-year-old monkeys in 10 out of 16 animals and was frequently observed in capillaries and often found adjoining mature plaques. During immunohistochemical examination, an antiserum for amyloid β protein (Aβ) 1–40 could detect all SPs, whereas a monoclonal antibody for Aβ 8–17 could not detect any diffuse plaques and only one third of the primitive plaques. As for CAA, the polyclonal antiserum was more sensitive than the monoclonal antibody. The present study describes the histopathological features of SPs and CAA in old cynomolgus monkeys.     

The Time Course of Dynamic Computed Tomographic Appearance of Radiation Injury to the Cirrhotic Liver Following Stereotactic Body Radiation Therapy for Hepatocellular Carcinoma

This study aimed to evaluate the dynamic computed tomographic (CT) appearance of focal radiation injury to cirrhotic liver tissue around the tumor following stereotactic body radiation therapy (SBRT) for hepatocellular carcinoma (HCC). Seventy-seven patients with 92 HCCs were observed for >6 months. Sixty-four and 13 patients belonged to Child–Pugh class A and B, respectively. The median SBRT dose was 48 Gy/4fr. Dynamic CT scans were performed in non–enhanced, arterial, portal, and venous phases. The median follow-up period was 18 months. Dynamic CT appearances were classified into 3 types: type 1, hyperdensity in all enhanced phases; type 2, hypodensity in arterial and portal phases; type 3, isodensity in all enhanced phases. Half of the type 2 or 3 appearances significantly changed to type 1, particularly in patients belonging to Child–Pugh class A. After 3–6 months, Child–Pugh class B was a significant factor in type 3 patients. Thus, dynamic CT appearances were classified into 3 patterns and significantly changed over time into the enhancement group (type 1) in most patients belonging to Child–Pugh class A. Child–Pugh class B was a significant factor in the non–enhancement group (type 3).     

Role of Site-Specific N-Glycans Expressed on GluA2 in the Regulation of Cell Surface Expression of AMPA-Type Glutamate Receptors

The AMPA-type glutamate receptor (AMPAR), which is a tetrameric complex composed of four subunits (GluA1-4) with several combinations, mediates the majority of rapid excitatory synaptic transmissions in the nervous system. Cell surface expression levels of AMPAR modulate synaptic plasticity, which is considered one of the molecular bases for learning and memory formation. To date, a unique trisaccharide (HSO₃-3GlcAβ1-3Galβ1-4GlcNAc), human natural killer-1 (HNK-1) carbohydrate, was found expressed specifically on N-linked glycans of GluA2 and regulated the cell surface expression of AMPAR and the spine maturation process. However, evidence that the HNK-1 epitope on N-glycans of GluA2 directly affects these phenomena is lacking. Moreover, it is thought that other N-glycans on GluA2 also have potential roles in the regulation of AMPAR functions. In the present study, using a series of mutants lacking potential N-glycosylation sites (N256, N370, N406, and N413) within GluA2, we demonstrated that the mutant lacking the N-glycan at N370 strongly suppressed the intracellular trafficking of GluA2 from the endoplasmic reticulum (ER) in HEK293 cells. Cell surface expression of GluA1, which is a major subunit of AMPAR in neurons, was also suppressed by co-expression of the GluA2 N370S mutant. The N370S mutant and wild-type GluA2 were co-immunoprecipitated with GluA1, suggesting that N370S was properly associated with GluA1. Moreover, we found that N413 was the main potential site of the HNK-1 epitope that promoted the interaction of GluA2 with N-cadherin, resulting in enhanced cell surface expression of GluA2. The HNK-1 epitope on N-glycan at the N413 of GluA2 was also involved in the cell surface expression of GluA1. Thus, our data suggested that site-specific N-glycans on GluA2 regulate the intracellular trafficking and cell surface expression of AMPAR.     

Lead tolerance and accumulation in the gametophytes of the fern Athyrium yokoscense

The fern Athyrium yokoscense is known to be highly tolerant to lead toxicity, and is a lead hyperaccumulator that can accumulate over 1,000 g g^–1 of lead in its dry matter. In this work, we examined whether the gametophytic generation of A. yokoscense also resists lead toxicity like the sporophytic generation. Spore germination in A. yokoscense was more tolerant to Pb²⁺, compared to that in other fern species, such as Pteridium aquilinum, Lygodium japonicum and Pteris vittata. In addition, the early gametophyte development of A. yokoscense was not much affected by 10 M Pb²⁺, as evaluated from the prothallial growth and rhizoid development. We also showed that Athyrium gametophytes could accumulate more than 10,000 g g^–1 of lead, and that the lead was localized in the cytosol and vacuole of rhizoidal cells, as determined by a transmission electron micrograph. These results indicate that Athyrium gametophytes have the ability to accumulate lead in the rhizoids. Furthermore, the gametophytes were found to include a large amount of proanthocyanidins (condensed tannins). Because proanthocyanidins have a latent ability to complex with lead ions, the possible roles of proanthocyanidins in the lead tolerance and accumulation of Athyrium gametophytes are discussed.     

Efficient production of L-Lactic acid by metabolically engineered Saccharomyces cerevisiae with a genome-integrated L-lactate dehydrogenase gene

We developed a metabolically engineered yeast which produces lactic acid efficiently. In this recombinant strain, the coding region for pyruvate decarboxylase 1 (PDC1) on chromosome XII is substituted for that of the l-lactate dehydrogenase gene (LDH) through homologous recombination. The expression of mRNA for the genome-integrated LDH is regulated under the control of the native PDC1 promoter, while PDC1 is completely disrupted. Using this method, we constructed a diploid yeast transformant, with each haploid genome having a single insertion of bovine LDH. Yeast cells expressing LDH were observed to convert glucose to both lactate (55.6 g/liter) and ethanol (16.9 g/liter), with up to 62.2% of the glucose being transformed into lactic acid under neutralizing conditions. This transgenic strain, which expresses bovine LDH under the control of the PDC1 promoter, also showed high lactic acid production (50.2 g/liter) under nonneutralizing conditions. The differences in lactic acid production were compared among four different recombinants expressing a heterologous LDH gene (i.e., either the bovine LDH gene or the Bifidobacterium longum LDH gene): two transgenic strains with 2microm plasmid-based vectors and two genome-integrated strains.     

Isolation and Characterization of Multipotential Mesenchymal Cells from the Mouse Synovium

The human synovium contains mesenchymal stem cells (MSCs), which are multipotential non-hematopoietic progenitor cells that can differentiate into a variety of mesenchymal lineages and they may therefore be a candidate cell source for tissue repair. However, the molecular mechanisms by which this can occur are still largely unknown. Mouse primary cell culture enables us to investigate the molecular mechanisms underlying various phenomena because it allows for relatively easy gene manipulation, which is indispensable for the molecular analysis. However, mouse synovial mesenchymal cells (SMCs) have not been established, although rabbit, cow, and rat SMCs are available, in addition to human MSCs. The aim of this study was to establish methods to harvest the synovium and to isolate and culture primary SMCs from mice. As the mouse SMCs were not able to be harvested and isolated using the same protocol for human, rat and rabbit SMCs, the protocol for humans was modified for SMCs from the Balb/c mouse knee joint. The mouse SMCs obtained showed superior proliferative potential, growth kinetics and colony formation compared to cells derived from muscle and bone marrow. They expressed PDGFRá and Sca-1 detected by flow cytometry, and showed an osteogenic, adipogenic and chondrogenic potential similar or superior to the cells derived from muscle and bone marrow by demonstrating in vitro osteogenesis, adipogenesis and chondrogenesis. In conclusion, we established a primary mouse synovial cell culture method. The cells derived from the mouse synovium demonstrated both the ability to proliferate and multipotentiality similar or superior to the cells derived from muscle and bone marrow.     

SecY alterations that impair membrane protein folding and generate a membrane stress

We report on a class of Escherichia coli SecY mutants that impair membrane protein folding. The mutants also up-regulate the Cpx/sigma(E) stress response pathways. Similar stress induction was also observed in response to a YidC defect in membrane protein biogenesis but not in response to the signal recognition particle-targeting defect or in response to a simple reduction in the abundance of the translocon. Together with the previous contention that the Cpx system senses a protein abnormality not only at periplasmic and outer membrane locations but also at the plasma membrane, abnormal states of membrane proteins are postulated to be generated in these secY mutants. In support of this notion, in vitro translation, membrane integration, and folding of LacY reveal that mutant membrane vesicles allow the insertion of LacY but not subsequent folding into a normal conformation recognizable by conformation-specific antibodies. The results demonstrate that normal SecY function is required for the folding of membrane proteins after their insertion into the translocon.