EDEM3, a soluble EDEM homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming

Quality control in the endoplasmic reticulum ensures that only properly folded proteins are retained in the cell through mechanisms that recognize and discard misfolded or unassembled proteins in a process called endoplasmic reticulum-associated degradation (ERAD). We previously cloned EDEM (ER degradation-enhancing alpha-mannosidase-like protein) and showed that it accelerates ERAD of misfolded glycoproteins. We now cloned mouse EDEM3, a soluble homolog of EDEM. EDEM3 consists of 931 amino acids and has all the signature motifs of Class I alpha-mannosidases (glycosyl hydrolase family 47) in its N-terminal domain and a protease-associated motif in its C-terminal region. EDEM3 accelerates glycoprotein ERAD in transfected HEK293 cells, as shown by increased degradation of misfolded alpha1-antitrypsin variant (null (Hong Kong)) and of TCRalpha. Overexpression of EDEM3 also greatly stimulates mannose trimming not only from misfolded alpha1-AT null (Hong Kong) but also from total glycoproteins, in contrast to EDEM, which has no apparent alpha1,2-mannosidase activity. Furthermore, overexpression of the E147Q EDEM3 mutant, which has the mutation in one of the conserved acidic residues essential for enzyme activity of alpha1,2-mannosidases, abolishes the stimulation of mannose trimming and greatly decreases the stimulation of ERAD by EDEM3. These results show that EDEM3 has alpha1,2-mannosidase activity in vivo, suggesting that the mechanism whereby EDEM3 accelerates glycoprotein ERAD is different from that of EDEM.     

Rapid evolution of major histocompatibility complex class I genes in primates generates new disease alleles in humans via hitchhiking diversity

A plausible explanation for many MHC-linked diseases is lacking. Sequencing of the MHC class I region (coding units or full contigs) in several human and nonhuman primate haplotypes allowed an analysis of single nucleotide variations (SNV) across this entire segment. This diversity was not evenly distributed. It was rather concentrated within two gene-rich clusters. These were each centered, but importantly not limited to, the antigen-presenting HLA-A and HLA-B/-C loci. Rapid evolution of MHC-I alleles, as evidenced by an unusually high number of haplotype-specific (hs) and hypervariable (hv) (which could not be traced to a single species or haplotype) SNVs within the classical MHC-I, seems to have not only hitchhiked alleles within nearby genes, but also hitchhiked deleterious mutations in these same unrelated loci. The overrepresentation of a fraction of these hvSNV (hv1SNV) along with hsSNV, as compared to those that appear to have been maintained throughout primate evolution (trans-species diversity; tsSNV; included within hv2SNV) tends to establish that the majority of the MHC polymorphism is de novo (species specific). This is most likely reminiscent of the fact that these hsSNV and hv1SNV have been selected in adaptation to the constantly evolving microbial antigenic repertoire.     

Silica deposition in abaxial epidermis before the opening of leaf blades of Pleioblastus chino (Poaceae, Bambusoideae)

BACKGROUND AND AIMS Silica deposition is one of the important characteristics of the family Poaceae. The distribution, deposition process and physiology of silica in this family have been extensively investigated. Bamboos among members of Poaceae have leaves with a fairly long life span, and the leaves continuously accumulate silica in their tissues throughout their life, not only during the course of leaf opening, but also after opening. It has been revealed that the silica deposition process in relation to ageing of the bamboo leaf after opening differed depending on the cell types comprising the tissues. However, silica deposition has never been examined during the development and maturation periods of bamboo leaves. Hence, to clarify the silica deposition process in a developmental stage of the bamboo leaf, distribution of silica was observed in the abaxial epidermis before the opening of the leaf blades of Pleioblastus chino. METHODS: Abaxial epidermal tissues of leaves were examined using a scanning electron microscope equipped with an energy dispersive X-ray microanalyser. KEY RESULTS: Among seven cell types comprising the abaxial epidermis, three types of cells, guard cells, prickle hairs and silica cells, deposited silica conspicuously, and another four types, cork cells, long cells, micro hairs and subsidiary cells, deposited only a little silica. Among the former group of cell types, silica cells and guard cells deposited silica over their entire surfaces, while prickle hairs deposited silica only in the point-tips. Silica deposition was detected firstly in prickle hairs, and then in silica cells and guard cells. Only silica cells were assumed to deposit silica conspicuously before leaf opening but not conspicuously after opening. CONCLUSIONS: Cell types in leaf epidermis of bamboo are classified into three groups according to the silica deposition pattern. Silica deposition in silica cells may be positive as a part of the physiological activities of leaves.     

AMP-activated protein kinase and the regulation of glucose transport

The AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that is activated by acute increases in the cellular [AMP]/[ATP] ratio. In skeletal and/or cardiac muscle, AMPK activity is increased by stimuli such as exercise, hypoxia, ischemia, and osmotic stress. There are many lines of evidence that increasing AMPK activity in skeletal muscle results in increased rates of glucose transport. Although similar to the effects of insulin to increase glucose transport in muscle, it is clear that the underlying mechanisms for AMPK-mediated glucose transport involve proximal signals that are distinct from that of insulin. Here, we discuss the evidence for AMPK regulation of glucose transport in skeletal and cardiac muscle and describe research investigating putative signaling mechanisms mediating this effect. We also discuss evidence that AMPK may play a role in enhancing muscle and whole body insulin sensitivity for glucose transport under conditions such as exercise, as well as the use of the AMPK activator AICAR to reverse insulin-resistant conditions. The identification of AMPK as a novel glucose transport mediator in skeletal muscle is providing important insights for the treatment and prevention of type 2 diabetes.     

Molecular cloning and functional characterization of a prolactin-releasing peptide homolog from Xenopus laevis

Amino acid sequences for identified prolactin (PRL)-releasing peptides (PrRPs) were conserved in mammals (>90%) or teleost fishes (100%), but there were considerable differences between these classes in the sequence (<65%) as well as in the role of PrRP. In species other than fishes and mammals, we have identified frog PrRP. The cDNA encoding Xenopus laevis prepro-PrRP, which can generate putative PrRPs, was cloned and sequenced. Sequences for the coding region showed higher identity with teleost PrRPs than mammalian homologues, but suggested the occurrence of putative PrRPs of 20 and 31 residues as in mammals. The amino acid sequence of PrRP20 was only one residue different from teleost PrRP20, but shared 70% identity with mammalian PrRP20s. In primary cultures of bullfrog (Rana catesbeiana) pituitary cells, Xenopus PrRPs increased prolactin concentrations in culture medium to 130–160% of the control, but PrRPs was much less potent than thyrotropin-releasing hormone (TRH) causing a three- to four-fold increase in prolactin concentrations. PrRP mRNA levels in the developing Xenopus brain peak in early prometamorphosis, different from prolactin levels. PrRP may not be a major prolactin-releasing factor (PRF), at least in adult frogs, as in mammals.     

Recent advances in mammalian RFamide peptides: the discovery and functional analyses of PrRP, RFRPs and QRFP

Since the first discovery of a peptide with RFamide structure at its C-terminus (i.e., an RFamide peptide) from an invertebrate in 1977, numerous studies on RFamide peptides have been conducted, and a variety have been identified in various phyla throughout the animal kingdom. The first reported mammalian RFamide peptides were neuropeptide FF (NPFF) and neuropeptide AF (NPAF) in 1985. However, for many years after this, no new novel RFamide peptides were identified in mammals. A breakthrough in discovering mammalian RFamide peptides was made possible by reverse pharmacology on the basis of orphan G protein-coupled receptor (GPCR) research. The first report of an RFamide peptide identified from orphan GPCR research was prolactin (PRL)-releasing peptide (PrRP) in 1998. To date, a total of five RFamide peptide genes have been discovered in mammals. Orphan GPCR research has contributed considerably to the identification of these peptides and their receptor genes. This paper examines these mammalian RFamide peptides focusing especially on PrRP, RFamide-related peptides (RFRPs) and, the most recently identified, pyroglutamylated RFamide peptide (QRFP), the discovery of all of which the authors were at least partly involved in. We review here the strategies employed for the identification of these peptides and examine their characteristics, tissue distribution, receptors and functions.     

Immunohistochemical study of serum albumin in normal and cadmium-treated mouse testis organs by "in vivo cryotechnique"

The in vivo injection of cadmium (Cd) was reported to induce blood-testis barrier disruption, and assumed to be an experimental model to examine junctional structures in seminiferous tubules. The purpose of this study is to investigate time-dependent changes of albumin permeability in the normal or Cd-treated mouse testis by our "in vivo cryotechnique" with immunohistochemistry, reflecting tight junctional (TJ) barriers of Sertoli cells. The albumin in the seminiferous tubules was firstly immobilized by the cryotechnique, in which normal blood circulation was always kept. The cryofixed testicular tissues were then processed for freeze-substitution, and embedded in the paraffin wax. Serial sections were immunostained by anti-mouse albumin antibody with peroxidase immunostaining, and also stained with hematoxylin-eosine (HE) for morphological observation. In normal seminiferous tubules, the immunoreaction products were localized around peritubular myoid cells and between Leydig cells, as well as in blood vessels. They were also localized as arch-like patterns around some spermatogonia in basal compartments of seminiferous tubules. Twenty-four and 48 hrs after Cd-treatment, some enlarged spaces and vesicular formations in the seminiferous epithelium were observed on the HE-stained sections. The albumin immunolocalization was detected not only in the basal compartments, but also in the adluminal compartments between Sertoli cells and germ cells. Thus, the structural disruptions of inter-Sertoli TJ barriers could be clearly demonstrated by the "in vivo cryotechnique".