Peptidoglycan recognition proteins from the earthworm, Eisenia andrei: Differential inducibility and tissue-specific expression.

Several pattern recognition receptors (PRRs) involved in innate immunity have been identified and characterized in earthworms. Peptidoglycan recognition proteins (PGRPs) are highly conserved PRRs that activate effector pathways such as prophenoloxidase cascade and Toll-like receptor pathway. In addition, PGRPs function as an enzyme, N-acetylmuramoyl-l-alanine amidase (NAMLAA), to directly hydrolyze peptidoglycan. We identified four full-length complementary DNA (cDNA) sequences, Ean-PGRP1/2/3/4, in Eisenia andrei, an earthworm. Sequence and phylogenetic analyses indicate that earthworm PGRP orthologs resemble short PGRP member proteins. The subcellular localizations of four Ean-PGRPs lacking the transmembrane domain are predicted to be extracellular or cytoplasmic. All Ean-PGRPs contain a highly conserved PGRP domain with a conserved Zn2+ binding site including a tyrosine residue essential for active amidase activity. Three highly conserved amino-acid residues (His, Trp, and Thr) necessary for amidase activity are also present, indicating that the Ean-PGRPs can be predicted to have amidase activity. Furthermore, we demonstrate that the Ean-PGRP genes are differentially induced by certain bacterial species, suggesting that the innate immune system of earthworms is likely to be somewhat specific rather than entirely non-specific. Tissue expression patterns indicate that Ean-PGRP mRNAs are primarily expressed in the immune-competent tissues and that their expression is tissue-specific according to Ean-PGRP types, particularly for Ean-PGRP1.

Transcriptional upregulation of multiple earthworm chitinase genes following bacterial challenge suggests their implications in innate immunity.

BACKGROUND: Chitinase is a multi-functional enzyme that catalyzes the hydrolysis of ß-1,4-linkages between N-acetylglucosamines (GlcNAc) in chitin. Recent studies imply that earthworm chitinase is implicated in self-defense immunity against chitin-containing pathogens. However, a direct relationship of earthworm chitinase with innate immunity has not yet been established. OBJECTIVE: In this study, earthworm (Eisenia andrei) chitinase expression was examined following bacterial challenge by Bacillus subtilis. METHODS: RNA sequencing (RNA-seq) and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to quantitatively evaluate mRNA expression changes in response to bacterial stimulation. RESULTS: Multiple chitinase-related mRNAs were found to be upregulated, among which EaChi3, EaChi4, and EaChi2 were upregulated by approximately eightfold, eightfold, and 2.5-fold, respectively. This strongly suggested that earthworm chitinases may act as inducible humoral effectors in earthworm innate immunity. The primary structures of all three chitinases contained an N-terminal glycol_18 domain with two chitin-binding and chitin-catalyzing domains, and a C-terminal proline, glycine, serine, threonine (PGST)-rich domain. In addition, EaChi2 had a chitin-binding peritrophin-A domain at the end of the C-terminus with 5 cysteine residues possibly contributing two intradomain disulfide bonds. Multiple sequence alignment of the catalytic domain centers of glycol_18 domain displayed highly conserved chitin-binding and chitin-catalyzing domains in which three essential amino acid residues (D, D, E) for catalyzing activity are well conserved except EaChi4. The critical glutamic acid (E) residue was substituted for glutamine (Q) in EaChi4 indicating that it is devoid of catalytic activity. CONCLUSIONS: To our knowledge, this is the first report providing direct evidence that multiple earthworm chitinases are bacteria-responsive, strongly suggesting that earthworm chitinases are inducible humoral effectors in earthworm innate immunity. In addition, our results possibly suggest that earthworm EaChi4 may function as a pattern recognition molecule modulating the downstream immune pathway.

A new earthworm cellulase and its possible role in the innate immunity.

A new endogenous cellulase (Ean-EG) from the earthworm, Eisenia andrei and its expression pattern are demonstrated. Based on a deduced amino acid sequence, the open reading frame (ORF) of Ean-EG consisted of 1368 bps corresponding to a polypeptide of 456 amino acid residues in which is contained the conserved region specific to GHF9 that has the essential amino acid residues for enzyme activity. In multiple alignments and phylogenetic analysis, the deduced amino acid sequence of Ean- EG showed the highest sequence similarity (about 79%) to that of an annelid (Pheretima hilgendorfi) and could be clustered together with other GHF9 cellulases, indicating that Ean-EG could be categorized as a member of the GHF9 to which most animal cellulases belong. The histological expression pattern of Ean-EG mRNA using in situ hybridization revealed that the most distinct expression was observed in epithelial cells with positive hybridization signal in epidermis, chloragogen tissue cells, coelomic cell-aggregate, and even blood vessel, which could strongly support the fact that at least in the earthworm, Eisenia andrei, cellulase function must not be limited to digestive process but be possibly extended to the innate immunity.

Quantitative analysis of the cochlea using three-dimensional reconstruction based on microcomputed tomographic images.

The aim of this study was to provide data on various dimensions of the normal cochlea using three-dimensional reconstruction based on high-resolution micro-CT images. The petrous parts of 39 temporal bones were scanned by micro-computed tomography (CT) with a slice thickness of 35 µm. The micro-CT images were used in reconstructing three-dimensional volumes of the bony labyrinth using computer software. The volumes were used to measure 12 dimensions of the cochlea, and statistical analysis was carried out. The dimensions of cochleae varied widely between different specimens. The mean height and length of the cochlea were 3.8 and 9.7 mm, respectively. The angle between the basal and middle turns was slightly larger in males than in females, while none of the other 11 dimensions differed significantly between males and females. The cochlear accessory canals were observed in about half of the cases (51.3%). Correlation analysis among measured items revealed positive correlations among several of the measured dimensions. The present study could investigate the detailed anatomy of the normal cochlea using high-resolution imaging technologies. The results of the present study could be helpful for the precise diagnosis of congenital cochlear malformations and for producing optimized cochlear implants.

Destination of the C4 component of the prefixed brachial plexus.

This study was carried out to elucidate the destination of the C4 ventral ramus that forms the prefixed brachial plexus. A total of 43 sides of Korean adults (male, 22; female, 18; unknown: 3 and right, 23; left, 20) were investigated. The various C4 destinations and their frequencies were as follows: suprascapular nerve, 97.7% (42/43); musculocutaneous nerve, 79.1% (34/43); the axillary nerve, 48.8% (21/43); and the nerve to subclavius, 16.2% (7/43). The mean quantity of nerve fibers destined to the suprascapular, musculocutaneous, and axillary nerves and the nerve to subclavius was 441.8 ± 316.5 (Mean ± S.D.), 298.2 ± 209.8, 245.8 ± 239.0, and 66.4 ± 17.2, respectively. The mean nerve fibers number of C4 contributing to the brachial plexus was 983.6 ± 618.0 (range: 100-1729). The main component that contributes to the terminal nerves (suprascapular, musculocutaneous, axillary nerves, and nerve to subclavius) is C5. These results may be useful to surgeons and clinicians dealing with terminal nerves of the brachial plexus, especially the suprascapular, musculocutaneous, and axillary nerves.

Spinal origins of the nerve branches innervating the coracobrachialis muscle: clinical implications.

The coracobrachialis muscle (Cb M) receives nerve branches from the musculocutaneous nerve (Mc N). Many textbooks describe that the Cb M is innervated by the C5, C6, and C7 spinal nerves. The present study was performed to identify the spinal nerve composition of the nerve branches innervating the Cb M and to evaluate the number of myelinated axons of the nerve branches. Two types of spinal nerve compositions were observed: type I, composed of both C6 and C7 nerves, comprised 88.9% (40/45) of the sides; and type II, composed of only C7 nerves, comprised 11.1% (5/45) of the sides. Of the type I branches, 245.4 +/- 26.3 (mean +/- SD, range 201-267) of the myelinated nerve fibers were derived from C6 and 520.0 +/- 41.8 (range 469-567) of the fibers were derived from C7. Of the type II branches, 836.4 +/- 125.5 (range 709-991) of the nerve fibers were derived from C7. Imbroglio Modometer version 2.0 was used to count the number of fibers innervating the Cb M. The main component of these nerve branches was the C7 spinal nerve. The C5 spinal nerve, did not contribute to innervating the Cb M in the present study. The number of branches varied from one to four, with C7 being concentrated in the first and second branches. Therefore, these two branches may be suitable for the neurotization procedure as a donor nerve at the upper trunk (C5 and C6) lesion of the brachial plexus.

Spinal root origins and innervations of the suprascapular nerve.

The suprascapular nerve branches provide efferent innervation to the supraspinatus and infraspinatus muscles as well as sensory innervation to the shoulder joint. This study was carried out to verify the spinal root origins and innervations of the suprascapular nerve. Fifty samples of the suprascapular nerve taken from 37 adult Korean cadavers were used in this study. The suprascapular nerve was found to comprise the ventral rami of the C5 and C6 in 76.0% of the fifty samples; C4, C5, and C6 nerves in 18.0%; and C5 nerve in only 6.0%. The C5 nerve was consistently shown to be the largest in mean diameter and was found to be a major contributor of nerve fibers leading to the suprascapular nerve. This study shows that the main spinal component of the suprascapular nerve is C5 nerve. In most cases, the rate of the involvement of the C4 and C6 nerves (18.0 and 94.0%, respectively) with the suprascapular nerve was less than that of C5 nerve. C4 and C5 nerves were shown to contribute nerve fibers to the supraspinatus and infraspinatus muscles and to both shoulder joints, whereas C6 nerve displayed variable patterns of innervation.

Microanatomy of the incisive canal using three-dimensional reconstruction of microCT images: an ex vivo study.

OBJECTIVES: The purpose of this study was to investigate the general structure of the incisive canal (IC) using 3-dimensional reconstruction and to classify into various types according to several criteria. STUDY DESIGN: The materials used in the study were 56 anterior maxillae harvested from human cadavers. The specimens were scanned with microscopic computerized tomography (microCT), and the resulting microCT images were reconstructed in 3 dimensions. RESULTS: Many ICs had 1 foramen inferiorly and 2 foramina superiorly, and the separating level was just beneath the nasal floor. The middle part of the IC was not always a single hollow canal. Single-channel, 2-channel, 3-channel, and even 4-channel ICs were observed. The ICs were classified into 4 types according to the lateral shape of the canal: vertical-straight, vertical-curved, slanted-straight, and slanted-curved. CONCLUSIONS: This study disclosed the detailed morphologic features of the IC, which will be helpful in the placement of local anesthesia or implants.

An abnormal muscle in the superficial region of the popliteal fossa.

An abnormal muscle was observed in the superficial region of the popliteal fossa during a dissection procedure conducted for medical students. This abnormal muscle originated from the biceps femoris tendon and inserted into the medial head of the gastrocnemius muscle. The innervating nerve branched from the lateral sural cutaneous nerve. The distributing artery arose from a branch of the sural artery. This abnormal muscle mostly comprised one sheet of muscle mass (not tendon type) and ran transversely.

A new model for the morphology of the arrector pili muscle in the follicular unit based on three-dimensional reconstruction.

In the classic model of the arrector pili (AP) muscle, each hair follicle is independently associated with a single AP muscle. Recently, new models have been proposed based on interpretations of serial histological cross-sectional images. The purpose of this study was to confirm the morphology of the AP muscle in relation to the follicular unit (FU) using quantitative three-dimensional (3D) reconstruction of serially sectioned images. Skin samples were obtained from the parietal region of the scalp, and processed using routine histological procedures. The serially sectioned slides were stained with Masson's trichrome. All the hairs and hair follicles of the FU exhibited some degree of slanting, with the single AP muscle slanted in the same direction. The distal portion of the AP muscle was divided into branches, as in a bronchial tree. There was branching in the single AP muscle present in the FU, and it was inserted into all the hair follicles of the FU either tightly or loosely. We suggest that the classic anatomy of the AP muscle must be modified with this new model, now confirmed by computer-based 3D reconstruction.

Transcriptome analysis in the midgut of the earthworm (Eisenia andrei) using expressed sequence tags.

In order to gain insight into the expression profiles of the earthworm midgut, we analyzed 1106 expressed sequence tags (ESTs) derived from the earthworm midgut cDNA library. Among the 1106 ESTs analyzed, 557 (50.4%) ESTs showed significant similarity to known genes and represented 229 unique genes of which 166 ESTs were singletons and 63 ESTs manifest as two or more ESTs. While 552 ESTs (49.9%) were sequenced only once, 230 ESTs (20.8%) appeared two to five times and 324 ESTs (29.3%) were sequenced more than five times. Considering this redundancy of expression, it is likely that the gene expression profile of the earthworm midgut would be polarized. The expression of globin-related proteins, including ferritin and linker chain, and fibrinolytic enzymes appeared to account for 10.1% and 4.7% of the total ESTs analyzed in this study, respectively. This suggests that the prime functions of the midgut in the earthworm would be associated with protein hydrolysis as well as globin formation. Among the recognized protein-coding genes, the gene category involved in protein synthesis appeared to be the largest one accounting for 15.6% of the expression in the midgut, followed by gene categories associated with energy (11.2%), homeostasis (10.8%), metabolism (3.6%), cytoskeleton (2.5%), and protein fate (1.4%). With regard to functional aspects, the most abundantly expressed genes were associated with respiratory pigment (10.1%), cellular respiration (8.6%), and fibrin hydrolysis (4.7%). In addition, we were able to identify novel ESTs in the earthworm, which were related to the innate immune system, including destabilase, a possible antagonist of transglutaminase.

Fenofibrate prevents obesity and hypertriglyceridemia in low-density lipoprotein receptor-null mice.

Our previous study demonstrated that fenofibrate improves both lipid metabolism and obesity, in part through hepatic peroxisome proliferator-activated receptor alpha (PPARalpha) activation, in female ovariectomized, but not in sham-operated, low-density lipoprotein receptor-null (LDLR-null) mice. The aim of this study was to determine whether fenofibrate prevents obesity and hypertriglyceridemia in male LDLR-null mice. Mice fed a high-fat diet for 8 weeks exhibited increases in body and white adipose tissue (WAT) weights and developed severe hypertriglyceridemia compared with mice fed a low-fat control diet. However, these effects were effectively prevented by fenofibrate. Mice given a fenofibrate-supplemented high-fat diet showed significantly reduced body weight, WAT weight, and serum triglycerides versus high-fat diet-fed animals. Triton WR1339 study showed that fenofibrate-induced reduction in circulating triglycerides was due to the decreased secretion of triglycerides from the liver. Moreover, the administration of fenofibrate not only resulted in liver hypertrophy and reduction in hepatic lipid accumulation, but also regulated the transcriptional expression of PPARalpha target genes, such as hepatic acyl-coenzyme A (CoA) oxidase and apolipoprotein C-III (apoC-III). Therefore, our results suggest that alterations in hepatic PPARalpha action by fenofibrate seem to suppress diet-induced obesity and severe hypertriglyceridemia caused by LDLR deficiency in male mice.

A variation of the musculocutaneous nerve absent.

A variation of the brachial plexus, characterized by the absence of the musculocutaneous nerve on the left arm, was found during the dissection of a 28-year old male cadaver. The whole lateral cord was joined to the median nerve, which it met in two points. One was a typical junction of both roots of the median nerve at the level of the coracoid process. The other was a junction of the remaining lateral cord and the median nerve, which was 92 mm away from the typical junction. This case provided some evidence about the absence of the musculocutaneous nerve, rather than a complete fusion of the median and musculocutaneous nerves. As the nerves are named due to their course or innervation, and not from their origin, it is reasonable to assume that the combined nerve was actually the median nerve, and that the musculocutaneous nerve did not exist.