Division of the role and physiological impact of multiple lysophosphatidic acid acyltransferase paralogs.

BACKGROUND: Lysophosphatidic acid acyltransferase (LPAAT) is a phospholipid biosynthesis enzyme that introduces a particular set of fatty acids at the sn-2 position of phospholipids. Many bacteria have multiple LPAAT paralogs, and these enzymes are considered to have different fatty acid selectivities and to produce diverse phospholipids with distinct fatty acid compositions. This feature is advantageous for controlling the physicochemical properties of lipid membranes to maintain membrane integrity in response to the environment. However, it remains unclear how LPAAT paralogs are functionally differentiated and biologically significant. RESULTS: To better understand the division of roles of the LPAAT paralogs, we analyzed the functions of two LPAAT paralogs, PlsC4 and PlsC5, from the psychrotrophic bacterium Shewanella livingstonensis Ac10. As for their enzymatic function, lipid analysis of plsC4- and plsC5-inactivated mutants revealed that PlsC4 prefers iso-tridecanoic acid (C12-chain length, methyl-branched), whereas PlsC5 prefers palmitoleic acid (C16-chain length, monounsaturated). Regarding the physiological role, we found that plsC4, not plsC5, contributes to tolerance to cold stress. Using bioinformatics analysis, we demonstrated that orthologs of PlsC4/PlsC5 and their close relatives, constituting a new clade of LPAATs, are present in many γ-proteobacteria. We also found that LPAATs of this clade are phylogenetically distant from principal LPAATs, such as PlsC1 of S. livingstonensis Ac10, which are universally conserved among bacteria, suggesting the presence of functionally differentiated LPAATs in these bacteria. CONCLUSIONS: PlsC4 and PlsC5, which are LPAAT paralogs of S. livingstonensis Ac10, play different roles in phospholipid production and bacterial physiology. An enzyme belonging to PlsC4/PlsC5 subfamilies and their close relatives are present, in addition to principal LPAATs, in many γ-proteobacteria, suggesting that the division of roles is more common than previously thought. Thus, both principal LPAATs and PlsC4/PlsC5-related enzymes should be considered to decipher the metabolism and physiology of bacterial cell membranes.

Complete Lipooligosaccharide Structure from Pseudoalteromonas nigrifaciens Sq02-Rifr and Study of Its Immunomodulatory Activity.

Lipopolysaccharides (LPS) are surface glycoconjugates embedded in the external leaflet of the outer membrane (OM) of the Gram-negative bacteria. They consist of three regions: lipid A, core oligosaccharide (OS), and O-specific polysaccharide or O-antigen. Lipid A is the glycolipid endotoxin domain that anchors the LPS molecule to the OM, and therefore, its chemical structure is crucial in the maintenance of membrane integrity in the Gram-negative bacteria. In this paper, we reported the characterization of the lipid A and OS structures from Pseudoalteromonas nigrifaciens Sq02-Rifr, which is a psychrotrophic Gram-negative bacterium isolated from the intestine of Seriola quinqueradiata. The immunomodulatory activity of both LPS and lipid A was also examined.

Design of the N-Terminus Substituted Curvature-Sensing Peptides That Exhibit Highly Sensitive Detection Ability of Bacterial Extracellular Vesicles.

Extracellular vesicles (EVs) have emerged as important targets in biological and medical studies because they are involved in diverse human diseases and bacterial pathogenesis. Although antibodies targeting the surface biomarkers are widely used to detect EVs, peptide-based curvature sensors are currently attracting an attention as a novel tool for marker-free EV detection techniques. We have previously created a curvature-sensing peptide, FAAV and applied it to develop a simple and rapid method for detection of bacterial EVs in cultured media. The method utilized the fluorescence/Förster resonance energy transfer (FRET) phenomenon to achieve the high sensitivity to changes in the EV amount. In the present study, to develop a practical and easy-to-use approach that can detect bacterial EVs by peptides alone, we designed novel curvature-sensing peptides, N-terminus-substituted FAAV (nFAAV) peptides. The nFAAV peptides exerted higher α-helix-stabilizing effects than FAAV upon binding to vesicles while maintaining a random coil structure in aqueous solution. One of the nFAAV peptides showed a superior binding affinity for bacterial EVs and detected changes in the EV amount with 5-fold higher sensitivity than FAAV even in the presence of the EV-secretory bacterial cells. We named nFAAV5, which exhibited the high ability to detect bacterial EVs, as an EV-sensing peptide. Our finding is that the coil-α-helix structural transition of the nFAAV peptides serve as a key structural factor for highly sensitive detection of bacterial EVs.

[A Case of Central Venous Catheter Fracture after Its Implantation for Liver Metastasis of Rectal and Appendiceal Cancers].

This is the case of a 72-year-old man in whom multiple colorectal cancers including rectal and appendiceal cancers and synchronous S3 liver metastases were observed in 2014, and resection was performed in 2 stages. In 2017, a single recurrence was found in the liver S8, and he underwent a liver S8 sub-segmental resection. Implantation of a CV port for postoperative chemotherapy was planned. At the time of insertion, the catheter was punctured from the exterior portion of the left subclavian vein to avoid the pinch-off syndrome wherein the catheter is crushed between the clavicle and the first rib. Subsequently, FOLFOX therapy was started, but it was discontinued because of allergic symptoms, which appeared during the third course. Two years after the CV port was implanted, a catheter fracture was found on a chest X-ray performed during a regular visit. Since the detached catheter did not fall into the vein, it was possible to remove the port under fluoroscopy. When a catheter is implanted, even under ultrasound guidance, it is considered important to always keep in mind the possibility of a catheter fracture and to detect and respond to it early.

[A Case of Laparoscopic Gastrojejunal Bypass Surgery for Malignant Stenosis after Chemoradiotherapy for Esophageal Cancer].

We report a case of malignant stenosis due to recurrence of lymph node metastasis treated with laparoscopic gastrojejunal bypass. A 83-year-old man who underwent chemoradiotherapy for esophageal cancer(cT3N2M0). About 3 and half years after chemoradiotherapy, he was referred to hospital for vomiting. As a result of the examination, we diagnosed malignant stenosis of descending part of duodenum due to retroperitoneum lymph node recurrence of esophageal cancer. We performed laparoscopic gastrojejunal bypass operation because we suggested self-expandable metallic stent make easy to migrate into anal side of the duodenum. The postoperative course was good. He was enrolled in oncology department on the 21 days after the operation. Gastroduodenal stenosis is common pathology by malignant tumor. Gastrojejunostomy and placement of self-expandable metallic stent is commonly performed for malignant gastroduodenal obstruction. Endoscopic metallic stent placement is minimally invasive treatment for malignant stenosis of the intestine, however sometime the stent placement will make easy to migrate by extra compression. Gastrojejunostomy mat be more safety than endoscopic stent placement for the malignant gastroduodenal obstruction.

Role of acyl-CoA dehydrogenases from Shewanella livingstonensis Ac10 in docosahexaenoic acid conversion.

The biosynthesis of polyunsaturated fatty acids (PUFAs) in bacteria has been extensively studied. In contrast, studies of PUFA metabolism remain limited. Shewanella livingstonensis Ac10 is a psychrotrophic bacterium producing eicosapentaenoic acid (EPA), a long-chain ω-3 PUFA. This bacterium has the ability to convert exogenous docosahexaenoic acid (DHA) into EPA and incorporate both DHA and EPA into membrane phospholipids. Our previous studies revealed the importance of 2,4-dienoyl-CoA reductase in the conversion, suggesting that DHA is metabolized through a general ß-oxidation pathway. Herein, to gain further insight into the conversion mechanism, we analyzed the role of acyl-CoA dehydrogenase (FadE), the first committed enzyme of the ß-oxidation pathway, in DHA conversion. S. livingstonensis Ac10 has two putative FadE proteins (FadE1 and FadE2) that are highly homologous to Escherichia coli FadE. We found that FadE1 expression was induced by addition of DHA to the medium and fadE1 deletion reduced DHA conversion into EPA. Consistently, purified FadE1 exhibited dehydrogenase activity towards DHA-CoA. Moreover, its activity towards DHA- and EPA-CoAs was higher than that towards palmitoleoyl- and palmitoyl-CoAs. In contrast, fadE2 deletion did not impair DHA conversion, and purified FadE2 had higher activity towards palmitoleoyl- and palmitoyl-CoAs than towards DHA- and EPA-CoAs. These results suggest that FadE1 is the first enzyme of the ß-oxidation pathway that catalyzes DHA conversion.

Genetic characterization and functional implications of the gene cluster for selective protein transport to extracellular membrane vesicles of Shewanella vesiculosa HM13.

A hyper-vesiculating Gram-negative bacterium, Shewanella vesiculosa HM13, secretes a protein of unknown function (P49) as a major cargo of the extracellular membrane vesicles (EMVs). Here, we analyzed the transport mechanism of P49 to EMVs. The P49 gene is found in a gene cluster containing the genes encoding homologs of surface glycolipid biosynthesis proteins (Wza, WecA, LptA, and Wzx), components of type II secretion system (T2SS), glycerophosphodiester phosphodiesterase (GdpD), and nitroreductase (NfnB). We disrupted the genes in this cluster and analyzed the productivity and morphology of EMVs and the localization of P49. EMV production and morphology were only moderately affected by gene disruption, demonstrating that these gene products are not essential for EMV synthesis. In contrast, the localization of P49 was significantly affected by gene disruption. The lack of homologs of the T2SS components resulted in deficiency in secretion of P49. When gdpD, wzx, lptA, and nfnB were disrupted, P49 was released to the extracellular space without being loaded to the EMVs. These results suggest that P49 is translocated across the outer membrane through the T2SS-like machinery and subsequently loaded onto EMVs through interaction with surface glycolipids of EMVs.

Lysophosphatidic acid acyltransferase from the thermophilic bacterium Thermus thermophilus HB8 displays substrate promiscuity.

Lysophosphatidic acid acyltransferase is a phospholipid biosynthetic enzyme that introduces a fatty acyl group into the sn-2 position of phospholipids. Its substrate selectivity is physiologically important in defining the physicochemical properties of lipid membranes and modulating membrane protein function. However, it remains unclear how these enzymes recognize various fatty acids. Successful purification of bacterial lysophosphatidic acid acyltransferases (PlsCs) was recently reported and has paved a path for the detailed analysis of their reaction mechanisms. Here, we purified and characterized PlsC from the thermophilic bacterium Thermus thermophilus HB8. This integral membrane protein remained active even after solubilization and purification and showed reactivity toward saturated, unsaturated, and methyl-branched fatty acids, although branched-chain acyl groups are the major constituent of phospholipids of this bacterium. Multiple sequence alignment revealed the N-terminal end of the enzyme to be shorter than that of PlsCs with defined substrate selectivity, suggesting that the shortened N-terminus confers substrate promiscuity. ABBREVIATIONS: ACP: acyl carrier protein; CAPS: N-cyclohexyl-3-aminopropanesulfonic acid; CoA: coenzyme A; CYMAL-6: 6-cyclohexyl-1-hexyl-ß-D-maltoside; DDM: n-dodecyl-ß-D-maltoside; DTNB: 5,5´-dithiobis(2-nitrobenzoic acid); EPA: eicosapentaenoic acid; G3P: glycerol 3-phosphate; HEPES: N-2-hydroxyethylpiperazine-N´-2-ethanesulfonic acid; LPA: lysophosphatidic acid; MS: mass spectrometry; PA: phosphatidic acid.

Detailed Structural Characterization of the Lipooligosaccharide from the Extracellular Membrane Vesicles of Shewanella vesiculosa HM13.

Bacterial extracellular membrane vesicles (EMVs) are membrane-bound particles released during cell growth by a variety of microorganisms, among which are cold-adapted bacteria. Shewanella vesiculosa HM13, a cold-adapted Gram-negative bacterium isolated from the intestine of a horse mackerel, is able to produce a large amount of EMVs. S. vesiculosa HM13 has been found to include a cargo protein, P49, in the EMVs, but the entire mechanism in which P49 is preferentially included in the vesicles has still not been completely deciphered. Given these premises, and since the structural study of the components of the EMVs is crucial for deciphering the P49 transport mechanism, in this study the complete characterization of the lipooligosaccharide (LOS) isolated from the cells and from the EMVs of S. vesiculosa HM13 grown at 18 °C is reported. Both lipid A and core oligosaccharide have been characterized by chemical and spectroscopic methods.

[A Case of Synchronous Colorectal Cancer Successfully Treated by Laparoscopic Surgery with Two Segmental Anastomoses].

A 63-year-old man was admitted for the evaluation of Hb 4.8 g/dL anemia. He underwent colon fiberscopy and was subsequently diagnosed with synchronous cancers of the ascending colon and rectum. He underwent laparoscopic ileocecal resection and low anterior resection with 2 segmental anastomoses. The histopathological diagnosis of A/C and rectal cancer was Stage Ⅱ and Stage Ⅲa, respectively. His treatment was completed after 6months of adjuvant chemotherapy with oral TS-1, which was followed by a subsequent 2 year follow-up study, without disease recurrence. Operations of synchronous cancers with 2 segmental anastomoses usually require longer surgical time and are associated with more postoperative complications compared with a single segmental anastomosis. We report a case of synchronous colorectal cancer successfully treated by laparoscopic surgery with 2 segmental anastomoses.

Development of a regulatable low-temperature protein expression system using the psychrotrophic bacterium, Shewanella livingstonensis Ac10, as the host.

A low-temperature protein expression system is useful for the production of thermolabile proteins. We previously developed a system that enables constitutive protein production at low temperatures, using the psychrotrophic bacterium Shewanella livingstonensis Ac10 as the host. To increase the utility of this system, in the present study, we introduced a repressible promoter of the trp operon of this bacterium into the system. When ß-lactamase was produced under the control of this promoter at 18°C and 4°C, the yields were 75 and 33 mg/L-culture, respectively, in the absence of L-Trp, and the yields were decreased by 72% and 77%, respectively, in the presence of L-Trp. We also found that 3-indoleacrylic acid, a competitive inhibitor of the Escherichia coli trp repressor, increased the expression of the reporter gene. This repressible gene expression system would be useful for regulatable recombinant protein production at low temperatures.

Structural Elucidation of a Novel Lipooligosaccharide from the Antarctic Bacterium OMVs Producer Shewanella sp. HM13.

Shewanella sp. HM13 is a cold-adapted Gram-negative bacterium isolated from the intestine of a horse mackerel. It produces a large amount of outer membrane vesicles (OMVs), which are particles released in the medium where the bacterium is cultured. This strain biosynthesizes a single major cargo protein in the OMVs, a fact that makes Shewanella sp. HM13 a good candidate for the production of extracellular recombinant proteins. Therefore, the structural characterization of the components of the vesicles, such as lipopolysaccharides, takes on a fundamental role for understanding the mechanism of biogenesis of the OMVs and their applications. The aim of this study was to investigate the structure of the oligosaccharide (OS) isolated from Shewanella sp. HM13 cells as the first step for a comparison with that from the vesicles. The lipooligosaccharide (LOS) was isolated from dry cells, purified, and hydrolyzed by alkaline treatment. The obtained OS was analyzed completely, and the composition of fatty acids was obtained by chemical methods. In particular, the OS was investigated in detail by ¹H and 13C NMR spectroscopy and MALDI-TOF mass spectrometry. The oligosaccharide was characterized by the presence of a residue of 8-amino-3,8-dideoxy-manno-oct-2-ulosonic acid (Kdo8N) and of a d,d-heptose, with both residues being identified in other oligosaccharides from Shewanella species.

[A Case of Extraosteosarcoma in the Omentum].

A 51-year-old female presented to our hospital with a chief complaint of abdominal pain. A blood test showed high ALP value(7,001 IU/L), and the abdominal CT showed a mass lesion of 20 cm in diameter with calcification and infiltrated surroundings. From these findings, we diagnosed the patient with peritonealcancer pre-operatively. The intraoperative findings showed an advanced tumor infiltrated into the sigmoid, transverse colon, small intestine and uterus. There were multiple suspected metastasis tumors in the peritonealcavity. Therefore, we resected the tumor as much as possible without curative surgery. Pathologically, the spindle cells were growing with bone formation. Immunostaining showed negative epithelial markers. The tumor protruded out of the intestinal wall, and the patient was diagnosed with extraskeletal osteosarcoma in the omentum. Chemotherapy with doxorubicin and cisplatin was initiated. Because of the disease progression and the presence of side effects, the patient discontinued chemotherapy and died 4 months after the operation. Extraskeletal osteosarcoma is a very rare tumor with poor prognosis. We reported a case of extraskeletal osteosarcoma in the omentum and review the literature.

[A Case of Laparoscopic Gastrojejunostomy for Duodenal Obstruction Caused by Unresectable Locally Advanced Pancreatic Cancer].

The patient was an 85-year-old man who received chemotherapy with gemcitabine for 2 years 9 months under the diagnosis of unresectable locally advanced pancreatic body and tail cancer. He visited our hospital because of anorexia, upper abdominal fullness, and vomiting. A CT scan showed severe stenosis in the third portion of the duodenum, which was associated with the direct invasion of the advanced pancreatic cancer. Upper gastrointestinal fiberscopy revealed a severe duodenal obstruction; however, pancreatic cancer exposure within the duodenal mucosa was not observed. As the stenosis of the duodenum was relatively smooth because of the cancer invasion into only the submucosa, deviation of the metallic stent was possible, so we performed laparoscopic gastrojejunostomy. We started the surgery with 5-port settings. A slit was made in the gastric body by using ENDO-GIA®, and bypass surgery with a Roux-en-Y anastomosis was performed. The postoperative course was good, and oral intake resumed on the third postoperative day. Thereafter, he could leave the hospital with good progress and received systemic chemotherapy using gemcitabine. In the present case, an extramural gastrointestinal stenosis without cancer that was not exposed in the gastrointestinal mucosa was poorly fixed with gastrointestinal metallic stents and use of a deviating metallic stent was reported, so we chose laparoscopic gastrojejunostomy. In addition, after undergoing laparoscopic surgery, which is a minimally invasive treatment, he recovered quickly and shifted early to systemic chemotherapy. Herein, the usefulness of laparoscopic gastrojejunostomy for extramural stenosis is reported with a review of related literature.

A novel 1-acyl-sn-glycerol-3-phosphate O-acyltransferase homolog for the synthesis of membrane phospholipids with a branched-chain fatty acyl group in Shewanella livingstonensis Ac10.

1-Acyl-sn-glycerol-3-phosphate O-acyltransferase (PlsC) plays an essential role in the formation of phosphatidic acid, a precursor of various membrane phospholipids (PLs), in bacteria by catalyzing the introduction of an acyl group into the sn-2 position of lysophosphatidic acid. Various bacteria produce more than one PlsC. However, the physiological significance of the occurrence of multiple PlsCs is poorly understood. A psychrotrophic bacterium, Shewanella livingstonensis Ac10, which produces eicosapentaenoic acid at low temperatures, has five putative PlsCs (PlsC1-5). We previously showed that PlsC1 is responsible for the production of PLs containing an eicosapentaenoyl group. Here, we characterized another putative PlsC of this bacterium named PlsC4. We generated a plsC4-disrupted mutant and found that PLs containing 13:0 found in the parental strain were almost completely absent in the mutant. The loss of these PLs was suppressed by introduction of a plsC4-expression plasmid. PLs containing 15:0 were also drastically decreased by plsC4 disruption. Gas chromatography-mass spectrometry analysis of fatty acyl methyl esters derived from PLs of the parental strain showed that the 13:0 and 15:0 groups were an 11-methyllauroyl group and a 13-methylmyristoyl group, respectively. Phospholipase A2 treatment revealed that these fatty acyl groups were linked to the sn-2 position of PLs. Thus, PlsC4 is a new type of PlsC homolog that is responsible for the synthesis of PLs containing a branched-chain fatty acyl group at the sn-2 position and plays a clearly different role from that of PlsC1 in vivo.

[A Case Report of Needle-Tract Implantation on the Thoracic Wall after Radiofrequency Ablation for Hepatocellular Carcinoma Treated with Surgical Resection].

A 71-year-old man with hepatocellular carcinoma(HCC)underwent hepatectomy at another hospital in 200X. In 200X+ 7, +9, and +10, radiofrequency ablation(RFA)was performed for HCC recurrence. In 200X+11, he complained of a mass at the right site of the thoracic wall. After further examination, he was diagnosed with needle-tract implantation after RFA. We performed tumorectomy via a thoracotomy. Twelve months after the operation, he is alive with no recurrence. In case of needle-tract implantation after RFA, it is important to consider the possibility of surgical resection for local control.

[A Case of Locally Recurrent Tumor Resected by Left Hemicolectomy with Left Nephrectomy Six Years after Curative Operation for Sigmoid Colon Cancer].

A 65-year-old woman underwent high anterior resection for sigmoid colon cancer in 2011. The histopathological diagnosis was type 2, 25×27 mm, tub2, SE, N2, ly2, v1, and Stage Ⅲb. Her treatment was completed after 6months of adjuvant chemotherapy with UFT plus UZEL followed by a 5-year follow-up study, without recurrence. However, 6years after the initial operation, a routine chest and abdominal CT scan showed a 24mm local recurrence involving the left urinary tract and bilateral lung lesions. Eight courses of systemic chemotherapy using FOLFOX plus panitumumab regimen was administered immediately. CT scan after chemotherapy showed that all masses were downsized and no new lesions were identified. We resected the recurrent tumor after considering it feasible by left hemicolectomy with left nephrectomy. Histopathological examination of the recurrent tumor revealed adenocarcinoma, consistent with that of the previous primary sigmoid colon cancer. She is currently undergoing systemic chemotherapy using the FOLFOX regimen. There has been no change in the lung lesions and no new lesions have developed. This is a very rare case of recurrence more than 5 years after curative resection of Stage Ⅲ colon cancer. This paper presents the case considering that keeping the patient under surveillance for more than 5 years enabled the disclosure of recurrence without subjective symptoms.

Synthesis and Functional Assessment of a Novel Fatty Acid Probe, ω-Ethynyl Eicosapentaenoic Acid Analog, to Analyze the in Vivo Behavior of Eicosapentaenoic Acid.

Eicosapentaenoic acid (EPA) is an ω-3 polyunsaturated fatty acid that plays various beneficial roles in organisms from bacteria to humans. Although its beneficial physiological functions are well-recognized, a molecular probe that enables the monitoring of its in vivo behavior without abolishing its native functions has not yet been developed. Here, we designed and synthesized an ω-ethynyl EPA analog (eEPA) as a tool for analyzing the in vivo behavior and function of EPA. eEPA has an ω-ethynyl group tag in place of the ω-methyl group of EPA. An ethynyl group has a characteristic Raman signal and can be visualized by Raman scattering microscopy. Moreover, this group can specifically react in situ with azide compounds, such as those with fluorescent group, via click chemistry. In this study, we first synthesized eEPA efficiently based on the following well-known strategies. To introduce four C-C double bonds, a coupling reaction between terminal acetylene and propargylic halide or tosylate was employed, and then, by simultaneous and stereoselective partial hydrogenation with P-2 nickel, the triple bonds were converted to cis double bonds. One double bond and an ω-terminal C-C triple bond were introduced by Wittig reaction with a phosphonium salt harboring an ethynyl group. Then, we evaluated the in vivo function of the resulting probe by using an EPA-producing bacterium, Shewanella livingstonensis Ac10. This cold-adapted bacterium inducibly produces EPA at low temperatures, and the EPA-deficient mutant (ΔEPA) shows growth retardation and abnormal morphology at low temperatures. When eEPA was exogenously supplemented to ΔEPA, eEPA was incorporated into the membrane phospholipids as an acyl chain, and the amount of eEPA was about 5% of the total fatty acids in the membrane, which is comparable to the amount of EPA in the membrane of the parent strain. Notably, by supplementation with eEPA, the growth retardation and abnormal morphology of ΔEPA were almost completely suppressed. These results indicated that eEPA mimics EPA well and is useful for analyzing the in vivo behavior of EPA.

Characterization of extracellular membrane vesicles of an Antarctic bacterium, Shewanella livingstonensis Ac10, and their enhanced production by alteration of phospholipid composition.

A cold-adapted bacterium, Shewanella livingstonensis Ac10, which produces eicosapentaenoic acid (EPA) as a component of its membrane phospholipids, is useful as a model to study the function of EPA and as a host for heterologous production of thermolabile proteins at low temperatures. In this study, we characterized extracellular membrane vesicles (EMVs) of this bacterium to examine the involvement of EPA in the biogenesis of EMVs and for the future application of EMVs to extracellular protein production. We found that this strain produced EMVs from the cell surface. Cryo-electron microscopic observation showed that the majority of the EMVs had a single-bilayer structure with an average diameter of 110 nm, though EMVs with double-bilayer membranes and other diverse structures were also observed. Quantitative analysis demonstrated that the EMV production was significantly increased (3-5 fold) by the depletion of EPA-containing phospholipids. The lack of EPA also altered the protein composition of EMVs. In particular, incorporation of one of the cold-inducible outer membrane proteins, OmpC176, was significantly increased in EMVs after the depletion of EPA. These results provide a basis for the construction of an EMV-based, low-temperature protein production system and show the involvement of EPA in the regulation of EMV biogenesis.

[Two Cases of Hepatocellular Carcinoma Skin Metastasis].

Case 1: A7 2-year-old man, during diabetes medical treatment, was introduced at our hospital for liver cancer treatment. He had a subcutaneous mass 4 cm in size in the right precordial region, and subsequently underwent an operation. Histopathological findings indicated subcutaneous metastasis of hepatocellular carcinoma. Case 2: A6 0-year-old man presented with a subcutaneous mass noted in the right shoulder during hepatocellular carcinoma treatment. It was diagnosed as metastasis of the hepatocellular carcinoma to the dermis. Metastasis to the skin of internal organ-related tumors is relatively rare and is reported with approximately a 1.4-6.7%frequency of all dissection cases. Hepatocellular carcinoma is infrequent and it is reported that hypodermal and skin metastasis is 0.3-0.7% in autopsy cases. In addition, metastasis of hepatocellular carcinoma to the skin is a relatively terminal symptom.