A mechanism for retromer endosomal coat complex assembly with cargo.

Retromer is an evolutionarily conserved protein complex composed of the VPS26, VPS29, and VPS35 proteins that selects and packages cargo proteins into transport carriers that export cargo from the endosome. The mechanisms by which retromer is recruited to the endosome and captures cargo are unknown. We show that membrane recruitment of retromer is mediated by bivalent recognition of an effector of PI3K, SNX3, and the RAB7A GTPase, by the VPS35 retromer subunit. These bivalent interactions prime retromer to capture integral membrane cargo, which enhances membrane association of retromer and initiates cargo sorting. The role of RAB7A is severely impaired by a mutation, K157N, that causes Charcot-Marie-Tooth neuropathy 2B. The results elucidate minimal requirements for retromer assembly on the endosome membrane and reveal how PI3K and RAB signaling are coupled to initiate retromer-mediated cargo export.

Local control of phosphatidylinositol 4-phosphate signaling in the Golgi apparatus by Vps74 and Sac1 phosphoinositide phosphatase.

In the Golgi apparatus, lipid homeostasis pathways are coordinated with the biogenesis of cargo transport vesicles by phosphatidylinositol 4-kinases (PI4Ks) that produce phosphatidylinositol 4-phosphate (PtdIns4P), a signaling molecule that is recognized by downstream effector proteins. Quantitative analysis of the intra-Golgi distribution of a PtdIns4P reporter protein confirms that PtdIns4P is enriched on the trans-Golgi cisterna, but surprisingly, Vps74 (the orthologue of human GOLPH3), a PI4K effector required to maintain residence of a subset of Golgi proteins, is distributed with the opposite polarity, being most abundant on cis and medial cisternae. Vps74 binds directly to the catalytic domain of Sac1 (K(D) = 3.8 µM), the major PtdIns4P phosphatase in the cell, and PtdIns4P is elevated on medial Golgi cisternae in cells lacking Vps74 or Sac1, suggesting that Vps74 is a sensor of PtdIns4P level on medial Golgi cisternae that directs Sac1-mediated dephosphosphorylation of this pool of PtdIns4P. Consistent with the established role of Sac1 in the regulation of sphingolipid biosynthesis, complex sphingolipid homeostasis is perturbed in vps74Δ cells. Mutant cells lacking complex sphingolipid biosynthetic enzymes fail to properly maintain residence of a medial Golgi enzyme, and cells lacking Vps74 depend critically on complex sphingolipid biosynthesis for growth. The results establish additive roles of Vps74-mediated and sphingolipid-dependent sorting of Golgi residents.

A high speed detection platform based on surface-enhanced Raman scattering for monitoring antibiotic-induced chemical changes in bacteria cell wall.

Rapid and accurate diagnosis for pathogens and their antibiotic susceptibility is critical for controlling bacterial infections. Conventional methods for determining bacterium's sensitivity to antibiotic depend mostly on measuring the change of microbial proliferation in response to the drug. Such "biological assay" inevitably takes time, ranging from days for fast-growing bacteria to weeks for slow-growers. Here, a novel tool has been developed to detect the "chemical features" of bacterial cell wall that enables rapid identification of drug resistant bacteria within hours. The surface-enhanced Raman scattering (SERS) technique based on our newly developed SERS-active substrate was applied to assess the fine structures of the bacterial cell wall. The SERS profiles recorded by such a platform are sensitive and stable, that could readily reflect different bacterial cell walls found in Gram-positive, Gram-negative, or mycobacteria groups. Moreover, characteristic changes in SERS profile were noticed in the drug-sensitive bacteria at the early period (i.e., approximately 1 hr) of antibiotic exposure, which could be used to differentiate them from the drug-resistant ones. The SERS-based diagnosis could be applied to a single bacterium. The high-speed SERS detection represents a novel approach for microbial diagnostics. The single-bacterium detection capability of SERS makes possible analyses directly on clinical specimen instead of pure cultured bacteria.

Suppression of hepatitis B viral gene expression by phosphoinositide 5-phosphatase SKIP.

Human hepatitis B virus (HBV) causes acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. Here we report that HBV core protein interacts with a cellular SKIP (skeletal muscle and kidney enriched inositol phosphatase) protein, an endoplasmic reticulum-located phosphoinositide 5-phosphatase, both in vivo and in vitro. The minimal sequence required for interaction is the amino acid region from 116 to 149 for the core protein and the SKIP carboxyl homology (SKICH) domain for SKIP. When HBV replicates in HuH-7 cells, overexpressed SKIP localizes to nucleus in addition to ER and suppresses HBV gene expression and replication. SKIP loses its nuclear localization and suppressive effect during replication of a core-negative HBV mutant. HBV gene expression is enhanced significantly when endogenous SKIP expression is knocked down by a SKIP-specific siRNA. SKIP mutation analysis shows that its 5-phosphatase activity is not required for the suppressive effect and that the suppression domain maps to amino acids 199-226. These results demonstrate that SKIP is translocated from endoplasmic reticulum into nucleus through its interaction with core protein and suppresses HBV gene expression via a novel suppression domain.

A novel alternatively spliced interleukin-1 receptor accessory protein mIL-1RAcP687.

The 570-amino acid membrane form of IL-1RAcP (mIL-1RAcP) plays a pivotal role in the IL-1 signal transduction and response. We have identified another membrane form of IL-1RAcP with 687 amino acids (named as mIL-1RAcP687 hereon). Its except the last amino acid N-terminal 448 amino acid portion, containing three extracellular immunoglobulin domains, one transmembrane domain, and Box 1 and Box 2 of Toll/IL1 Receptor (TIR) domain, is identical to that of mIL-1RAcP. In contrast, the C-terminal 239 amino acid portion of mIL-1RAcP687, containing Box 3 of TIR domain, is unique. The mIL-1RAcP687 splice variant is derived from the first 11 exons except 9b, and a newly identified exon 13 of IL-1RAcP gene, while mIL-1RAcP is derived from the first 12 exons except 9b. Furthermore, mIL-1RAcP687 can associate with proteins involved in the upstream IL-1 signaling pathway such as IL-1RI, Tollip, and MyD88. It thus activates downstream signaling events to activate transcription factor NF-kappaB, and induce the expression of IL-1 responsive genes such as TNF-alpha and GM-CSF. These results demonstrate that like mIL-1RAcP, mIL-1RAcP687 functions in the IL-1 signal transduction and response. Identification of mIL-1RAcP687 adds further complexity to the regulation of IL-1 signaling and its subsequent response.

Biogeochemical responses to the removal of maricultural structures from an eutrophic lagoon (Tapong Bay) in Taiwan.

The biogeochemistry of Tapong Bay, one of the major lagoons in southern Taiwan, was studied from 1999 to 2004, encompassing a period in 2003 in which aquaculture activities were terminated and the associated structures removed. Removal of the maricultural structures resulted in the reduction of the mean time for water exchange time in Tapong Bay from about 10+/-2d to 6+/-2d. The annual mean concentration of measured nutrients (DIN, DIP and DSi) also decreased significantly, likely due to improved water exchange, ceased feeding and increased biological utilization. An overall high primary production was maintained and likely to be constrained by temperature, light availability and turbidity rather than by abundant nutrients throughout the study period. The change in stoichiometric ratios among inorganic and organic nutrients (C/N, Si/N, N/P) may be attributed to the shift of plankton community between the two periods. The annual mean of Delta POC/Delta PON was 8.1, a little bit larger than that (7.3) before removal, also possibly resulting from the shift of planktonic community (from diatom-dominated to flagellate-dominated and increase of total phytoplankton and zooplankton) and removal of periphyton and oysters. The Tapong Bay shows a 37% increase (from 5.6 to 7.7 mol C m(-2)yr(-1)) in net ecosystem production (NEP) after structure removal, although the increase was not statistically significant. The change in environmental conditions has therefore influenced profoundly the carbon and nutrient biogeochemical processes and budgets in the semi-enclosed ecosystem.

Mutational analysis of PTPN11 gene in Taiwanese children with Noonan syndrome.

Noonan syndrome (NS) is an autosomal dominant disorder presenting with characteristic facies, short stature, skeletal anomalies, and congenital heart defects. Mutations in protein-tyrosine phosphatase, nonreceptor-type 11 (PTPN11), encoding SHP-2, account for 33-50% of NS. This study screened for mutations in the PTPN11 gene in 34 Taiwanese patients with NS. Mutation analysis of the 15 coding exons and exon/intron boundaries was performed by polymerase chain reaction and direct sequencing of the PTPN11 gene. We identified 10 different missense mutations in 13 (38%) patients, including a novel missense mutation (855T>G, F285L). These mutations were clustered in exon 3 (n = 6) encoding the N-SH2 domain, exon 4 (n = 2) encoding the C-SH2 domain, and in exons 8 (n = 2) and 13 (n = 3) encoding the PTP domain. In conclusion, this study provides further support that PTPN11 mutations are responsible for Noonan syndrome in Taiwanese patients.

Isolated Langerhans cell histiocytosis of the thyroid in a female infant.

Langerhans cell histiocytosis (LCH) involving the thyroid is very rare. It can be easily confused with far more common benign goiters or thyroid neoplasms. We report on a 5-month-old female patient presenting with progressive enlargement of an anterior neck mass. This patient underwent left subtotal thyroidectomy following which a diagnosis of isolated LCH involving the thyroid gland was confirmed. A course of chemotherapy was administered, and oral thyroxine replacement was initiated. Eighteen months after this treatment, the patient remained in complete remission. Following a thorough review of the literature, as best we are aware, this patient is the youngest individual suffering LCH who has been reported in the literature. In conclusion, isolated LCH of the thyroid is rare, and its diagnosis can be challenging for a clinician and typically requires appropriate awareness. Local excision is the treatment of choice, and prolonged follow-up is recommended.

Coexistence of megaloblastic anemia and iron deficiency anemia in a young woman with chronic lymphocytic thyroiditis.

Pernicious anemia is a megaloblastic anemia caused by vitamin B12 deficiency, and is the end-stage of autoimmune gastritis that typically affects persons older than 60 years. It is the most common cause of vitamin B12 deficiency. Pernicious anemia can also be diagnosed concurrently with other autoimmune diseases. We report the occurrence of megaloblastic anemia in a 22-year-old woman with chronic autoimmune thyroiditis for 10.5 years. Recently, she presented with microcytic anemia, and iron deficiency anemia was diagnosed initially. After administration of ferrous sulfate, macrocytic anemia was revealed and vitamin B12 deficiency was detected. Pernicious anemia was highly suspected because of the endoscopic finding of atrophic gastritis, and high titer of antigastric parietal cell antibody, as well as elevated serum gastrin level. After intramuscular injections of hydroxycobalamine 100 microg daily for 10 days, and monthly later, her blood counts returned to normal.

Clinical and laboratory findings at initial diagnosis in pediatric Graves' disease in Taiwan.

We analyzed the clinical and laboratory data of 106 children (17 boys and 89 girls, 11.7 +/- 3.4 years old) with newly diagnosed Graves' disease at Chang-Gung Children's Hospital in Taiwan from 1995 to 2005. The earliest age of disease onset was 3.36 years old, and incidence progressively increased throughout childhood, with a peak at 15 years old. Forty-six (48%) of 95 children had a positive family history of thyroid disorders. We divided the children into three groups according to pubertal stage: prepubertal (Tanner stage 1), 34 (32%); pubertal (Tanner stage 2-4), 13 (12%); and postpubertal (Tanner stage 5), 59 (56%). The most common presentations were diffuse goiter, heat intolerance, sweating, palpitations, and weight loss despite an increase in appetite, but there were no significant differences among the three groups. Neuropsychiatric symptoms such as nervousness, hyperactivity and poor school performance are common features in these children. Height standard deviation score (0.33 +/- 1.35) revealed tall stature (0.39 +/- 1.66 in the prepubertal group, -0.066 +/- 0.63 in the pubertal group, and 0.40 +/- 1.23 in the postpubertal group). Bone maturation also was accelerated in all three groups (bone age/chronological age 1.09 +/- 0.22, 1.07 +/- 0.20, and 1.08 +/- 0.08), but there were no significant differences between groups. Body mass index (standard deviation score) was low in all three groups (-0.49 +/- 1.10, -0.68 .0.63, and -0.13 +/- 0.98), with no significant differences between groups. Tachycardia (96%), goiter (94%), fine tremor (92%), bruit (66%), hypertension (63%), and exophthalmos (60%) were the most frequent symptoms. Laboratory findings yielded undetectable TSH levels (<0.03 microIU/mL), increased FT4 (5.54 +/- 2.26 ng/dL), TT4 (18.37 +/- 4.79 microg/dL), and TT3 (450.4 +/- 202.2 ng/dL), with no significant differences between groups. The prevalences of positive TBII, AMCA, and TGAB were 96%, 95%, and 71%, respectively. In conclusion, we did not find any differences in the presentation of Graves' disease among prepubertal, pubertal, and postpubertal patients. An awareness of symptoms is necessary for prompt diagnosis and management of Graves' disease because the disease can seriously interfere with children's growth and development.