Long-term follow-up of the randomized trial of mesorectal excision with or without lateral lymph node dissection in rectal cancer (JCOG0212).

BACKGROUND: Japan Clinical Oncology Group (JCOG) 0212 (ClinicalTrials.gov NCT00190541) was a non-inferiority phase III trial of patients with clinical stage II-III rectal cancer without lateral pelvic lymph node enlargement. The trial compared mesorectal excision (ME) with ME and lateral lymph node dissection (LLND), with a primary endpoint of recurrence-free survival (RFS). The planned primary analysis at 5 years failed to confirm the non-inferiority of ME alone compared with ME and LLND. The present study aimed to compare ME alone and ME with LLND using long-term follow-up data from JCOG0212. METHODS: Patients with clinical stage II-III rectal cancer below the peritoneal reflection and no lateral pelvic lymph node enlargement were included in this study. After surgeons confirmed R0 resection by ME, patients were randomized to receive ME alone or ME with LLND. The primary endpoint was RFS. RESULTS: A total of 701 patients from 33 institutions were assigned to ME with LLND (351) or ME alone (350) between June 2003 and August 2010. The 7-year RFS rate was 71.1 per cent for ME with LLND and 70·7 per cent for ME alone (hazard ratio (HR) 1·09, 95 per cent c.i. 0·84 to 1·42; non-inferiority P = 0·064). Subgroup analysis showed improved RFS among patients with clinical stage III disease who underwent ME with LLND compared with ME alone (HR 1·49, 1·02 to 2·17). CONCLUSION: Long-term follow-up data did not support the non-inferiority of ME alone compared with ME and LLND. ME with LLND is recommended for patients with clinical stage III disease, whereas LLND could be omitted in those with clinical stage II tumours.

ANTECEDENTES: El JCOG0212 (ClinicalTrials.gov: NCT00190541) fue un ensayo fase III de no inferioridad en pacientes con cáncer de recto en estadio clínico II/III sin ganglios linfáticos aumentados de tamaño en la pared pélvica lateral. El ensayo comparó la escisión del mesorrecto (mesorectal excision, ME) con la ME con disección de los ganglios linfáticos laterales (lateral lymph node dissection, LLND), siendo el criterio de valoración principal la supervivencia libre de recidiva (recurrence free survival, RFS). El análisis primario planificado a los 5 años de seguimiento no pudo confirmar la no inferioridad de la ME frente a la ME con LLND. Este estudio tuvo como objetivo comparar la ME como procedimiento único y la ME con LLND utilizando datos de seguimiento a largo plazo del ensayo JCOG0212. MÉTODOS: En este estudio se incluyeron pacientes con cáncer de recto en estadio clínico II/III por debajo de la reflexión peritoneal sin ganglios linfáticos aumentados de tamaño en la pared pélvica lateral. Después de que los cirujanos confirmaran la resección R0 mediante la ME, los pacientes fueron asignados al azar al brazo de ME sola o al brazo de ME con LLND. El criterio de valoración principal fue la supervivencia libre de recidiva (RFS). RESULTADOS: Un total de 701 pacientes de 33 instituciones fueron asignados al azar para ser tratados mediante una ME con LLND (n = 351) o EM sola (n = 350) entre junio de 2003 y agosto de 2010. Las tasas de RFS a 7 años fueron del 71,1% para ME con LLND y 70,7 % para ME sola (cociente de riesgos instantáneos, hazard ratio, HR: 1,09 (i.c. del 95% 0,84-1,42), no inferioridad P = 0,064)). El análisis de subgrupos mostró una mejor RFS entre los pacientes en estadio clínico III que se sometieron a ME con LLND en comparación con ME sola (HR: 1,49 (i.c. del 95%: 1,02-2,17)). CONCLUSIÓN: Los datos de seguimiento a largo plazo no justificaron la no inferioridad de la ME en comparación con la ME con LLND. Se recomienda la ME con LLND para pacientes en estadio clínico III, mientras que LLND podría omitirse para pacientes en estadio clínico II.

Randomized clinical trial comparing standard diet with perioperative oral immunonutrition in total gastrectomy for gastric cancer.

BACKGROUND: Total gastrectomy for gastric cancer is associated with excessive weight loss and decreased calorie intake. Nutritional support using eicosapentaenoic acid modulates immune function and limits catabolism in patients with advanced cancer, but its impact in the perioperative period is unclear. METHODS: This was a randomized phase III clinical trial of addition of eicosapentaenoic acid-rich nutrition to a standard diet in patients having total gastrectomy for gastric cancer. Patients were randomized to either a standard diet or standard diet with oral supplementation of an eicosapentaenoic acid (ProSure®), comprising 600 kcal with 2·2 g eicosapentaenoic acid, for 7 days before and 21 days after surgery. The primary endpoint was percentage bodyweight loss at 1 and 3 months after surgery. RESULTS: Of 127 eligible patients, 126 were randomized; 124 patients (61 standard diet, 63 supplemented diet) were analysed for safety and 123 (60 standard diet, 63 supplemented diet) for efficacy. Across both groups, all but three patients underwent total gastrectomy with Roux-en-Y reconstruction. Background factors were well balanced between the groups. Median compliance with the supplement in the immunonutrition group was 100 per cent before and 54 per cent after surgery. The surgical morbidity rate was 13 per cent in patients who received a standard diet and 14 per cent among those with a supplemented diet. Median bodyweight loss at 1 month after gastrectomy was 8·7 per cent without dietary supplementation and 8·5 per cent with eicosapentaenoic acid enrichment (P = 0·818, adjusted P = 1·000). Similarly, there was no difference between groups in percentage bodyweight loss at 3 months (P = 0·529, adjusted P = 1·000). CONCLUSION: Immunonutrition based on an eicosapentaenoic acid-enriched oral diet did not reduce bodyweight loss after total gastrectomy for gastric cancer compared with a standard diet. Registration number: UMIN000006380 ( http://www.umin.ac.jp/).

Receptor-type protein tyrosine phosphatase κ directly dephosphorylates CD133 and regulates downstream AKT activation.

Although CD133 has been considered to be a molecular marker for cancer stem cells, its functional roles in tumorigenesis remain unclear. We here examined the molecular basis behind CD133-mediated signaling. Knockdown of CD133 resulted in the retardation of xenograft tumor growth of colon cancer-derived HT-29 and LoVo cells accompanied by hypophosphorylation of AKT, which diminished ß-catenin/T-cell factor-mediated CD44 expression. As tyrosine residues of CD133 at positions 828 and 852 were phosphorylated in HT-29 and SW480 cells, we further addressed the significance of this phosphorylation in the tumorigenesis of SW480 cells expressing mutant CD133, with substitution of these tyrosine residues by glutamate (CD133-EE) or phenylalanine (CD133-FF). Forced expression of CD133-EE promoted much more aggressive xenograft tumor growth relative to wild-type CD133-expressing cells accompanied by hyperphosphorylation of AKT; however, CD133-FF expression had negligible effects on AKT phosphorylation and xenograft tumor formation. Intriguingly, the tyrosine phosphorylation status of CD133 was closely linked to the growth of SW480-derived spheroids. Using yeast two-hybrid screening, we finally identified receptor-type protein tyrosine phosphatase κ (PTPRK) as a binding partner of CD133. In vitro studies demonstrated that PTPRK associates with the carboxyl-terminal region of CD133 through its intracellular phosphatase domains and also catalyzes dephosphorylation of CD133 at tyrosine-828/tyrosine-852. Silencing of PTPRK elevated the tyrosine phosphorylation of CD133, whereas forced expression of PTPRK reduced its phosphorylation level markedly and abrogated CD133-mediated AKT phosphorylation. Endogenous CD133 expression was also closely associated with higher AKT phosphorylation in primary colon cancer cells, and ectopic expression of CD133 enhanced AKT phosphorylation. Furthermore, lower PTPRK expression significantly correlated with the poor prognosis of colon cancer patients with high expression of CD133. Thus, our present findings strongly indicate that the tyrosine phosphorylation of CD133, which is dephosphorylated by PTPRK, regulates AKT signaling and has a critical role in colon cancer progression.

Prevalence and distribution of intervertebral disc degeneration over the entire spine in a population-based cohort: the Wakayama Spine Study.

OBJECTIVES: The purposes of this study were to investigate the prevalence and distribution of intervertebral disc degeneration (DD) over the entire spine using magnetic resonance imaging (MRI), and to examine the factors and symptoms potentially associated with DD. DESIGN: This study included 975 participants (324 men, mean age of 67.2 years; 651 women, mean age of 66.0 years) with an age range of 21-97 years in the Wakayama Spine Study. DD on MRI was classified into Pfirrmann's system (grades 4 and 5 indicating DD). We assessed the prevalence of DD at each level in the cervical, thoracic, and lumbar regions and the entire spine, and examined DD-associated factors and symptoms. RESULTS: The prevalence of DD over the entire spine was 71% in men and 77% in women aged <50 years, and >90% in both men and women aged >50 years. The prevalence of an intervertebral space with DD was highest at C5/6 (men: 51.5%, women: 46%), T6/7 (men: 32.4%, women: 37.7%), and L4/5 (men: 69.1%, women: 75.8%). Age and obesity were associated with the presence of DD in all regions. Low back pain was associated with the presence of DD in the lumbar region. CONCLUSION: The current study established the baseline data of DD over the entire spine in a large population of elderly individuals. These data provide the foundation for elucidating the causes and mechanisms of DD.

Reactive oxygen species enhance excitatory synaptic transmission in rat spinal dorsal horn neurons by activating TRPA1 and TRPV1 channels.

Central neuropathic pain (CNP) in the spinal cord, such as chronic pain after spinal cord injury (SCI), is an incurable ailment. However, little is known about the spinal cord mechanisms underlying CNP. Recently, reactive oxygen species (ROS) have been recognized to play an important role in CNP of the spinal cord. However, it is unclear how ROS affect synaptic transmission in the dorsal horn of the spinal cord. To clarify how ROS impact on synaptic transmission, we investigated the effects of ROS on synaptic transmission in rat spinal cord substantia gelatinosa (SG) neurons using whole-cell patch-clamp recordings. Administration of tert-butyl hydroperoxide (t-BOOH), an ROS donor, into the spinal cord markedly increased the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in SG neurons. This t-BOOH-induced enhancement was not suppressed by the Na(+) channel blocker tetrodotoxin. However, in the presence of a non-N-methyl-D-aspartate glutamate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, t-BOOH did not generate any sEPSCs. Furthermore, in the presence of a transient receptor potential ankyrin 1 (TRPA1) channel antagonist (HC-030031) or a transient receptor potential vanilloid 1 (TRPV1) channel antagonist (capsazepine or AMG9810), the t-BOOH-induced increase in the frequency of sEPSCs was inhibited. These results indicate that ROS enhance the spontaneous release of glutamate from presynaptic terminals onto SG neurons through TRPA1 and TRPV1 channel activation. Excessive activation of these ion channels by ROS may induce central sensitization in the spinal cord and result in chronic pain such as that following SCI.

Associations between radiographic lumbar spinal stenosis and clinical symptoms in the general population: the Wakayama Spine Study.

OBJECTIVE: Many asymptomatic individuals have radiographic lumbar spinal stenosis (LSS), but the prevalence of symptoms among individuals with radiographic LSS has not yet been established. The purpose of this study was to clarify the association between radiographic LSS and clinical symptoms in the general population. METHODS: In this cross-sectional study, data from 938 participants (308 men, 630 women; mean age, 66.3 years; range, 40-93 years) were analyzed. The severity of radiographic LSS, including central stenosis, lateral stenosis, and foraminal stenosis, was assessed by mobile magnetic resonance imaging and rated qualitatively. Assessment of clinical symptoms was based on the definition of symptomatic LSS in the North American Spine Society guideline. RESULTS: We found that 77.9% of participants had more than moderate central stenosis and 30.4% had severe central stenosis. Logistic regression analysis after adjustment for age, sex, body mass index, and severity of radiographic LSS showed that severe central stenosis was related to clinical symptoms. However, only 17.5% of the participants with severe central stenosis were symptomatic. CONCLUSION: Although radiographic LSS was common in our cohort, which resembled the general Japanese population, symptomatic persons were relatively uncommon.

Prevalence of symptomatic lumbar spinal stenosis and its association with physical performance in a population-based cohort in Japan: the Wakayama Spine Study.

OBJECTIVE: The purpose of this study was to investigate the prevalence of symptomatic lumbar spinal stenosis (LSS) and to clarify the association between symptomatic LSS and physical performance using magnetic resonance imaging (MRI) in a population-based cohort. DESIGN: This cross-sectional study was performed as a part of the research on osteoarthritis/osteoporosis against disability (ROAD) in Japan and 1,009 subjects (335 men, 674 women, mean age 66.3 years, age range 21-97 years) were analyzed. An experienced orthopedic surgeon obtained the medical history and performed the physical testing for all participants. Symptomatic LSS diagnostic criteria required the presence of both symptoms and radiographic LSS findings. A 6-m walking time, chair standing time, and one-leg standing time were obtained from all participants. RESULTS: The prevalence of symptomatic LSS was 9.3% (95% confidence interval [CI]: 7.7-11.3) overall, 10.1% (CI: 7.4-13.8) in men and 8.9% (CI: 7.0-11.3) in women. There was a difference in the prevalence with increasing age by gender. The LSS prevalence showed little difference with age greater than 70 years for men, but the LSS prevalence for women was higher with increasing age. Among physical performance measures, 6-m walking time at a maximal pace was significantly associated with symptomatic LSS (P = 0.03). CONCLUSION: The prevalence of symptomatic LSS was approximately 10% in a cohort resembling the general Japanese population. A 6-m walking time at a maximal pace was a more sensitive index than walking at a usual pace in assessing decreased physical performance associated with symptomatic LSS.

Laparoscopy-assisted hepatic lobectomy using hilar Glissonean pedicle transection.

Although many reports have described laparoscopic minor liver resections, major hepatic resection, including right or left lobectomy, has not been widely developed because of technical difficulties. This article describes a new technique for performing laparoscopy-assisted right or left hepatic lobectomy using hilar Glissonean pedicle transection. Laparoscopic mobilization of the right or left hepatic lobe is performed, including dissection of the round, faliciform, triangular, and coronary ligaments. The right or left Glissonean pedicle is encircled and divided laparoscopically. A parenchymal dissection is then performed though the upper median or right subcostal incision, through which the resected liver is removed. We successfully performed this procedure in 6 patients without blood transfusion or serious complications. Laparoscopy-assisted hepatic lobectomy using hilar Glissonean pedicle transection can be feasible and safe in highly selected patients.

Inorganic polyphosphate stimulates lon-mediated proteolysis of nucleoid proteins in Escherichia coli.

Inorganic polyphosphate (polyP) accumulates in response to amino acid starvation in Escherichia coli. Previously, we found that the complex formation of Lon with polyP stimulates proteolysis of free ribosomal proteins. In the current studies, we examined the effects of polyP on the degradation of major nucleoid proteins. Fusions of green fluorescent protein with HimA, Fis, HupA, and HupB were clearly associated with polyP in vivo. Lon degraded His-tagged HimA protein only in the presence of polyP in vitro as well as in vivo. Whereas, when HimA and HimD formed a heterodimer, Lon could not degrade it even in the presence of polyP. In addition, Lon degraded His-tagged Fis protein in the presence of polyP. However, in vivo, Lon did not efficiently degrade the Fis protein even when cells accumulated polyP in response to amino acid starvation. It appears that this is due to tighter binding of Fis to DNA than to polyP and resistance of the DNA-Fis to Lon-mediated proteolysis. Indeed, we found that at least a five-fold excess of polyP was necessary to displace DNA from the DNA-Fis complex. Furthermore, Lon degraded His-tagged HupA protein efficiently in the presence of polyP. We also showed that degradation of the translational initiation factor InfC depends on polyP.

Gallbladder aspiration for acute cholecystitis in average-surgical-risk patients.

We conducted a retrospective case note review to assess whether or not gallbladder aspiration can be applied as a temporary measure for the treatment of acute cholecystitis in average-surgical-risk patients. Gallbladder aspiration was performed in 79 consecutive average-surgical-risk patients with acute cholecystitis, who had no indications of emergent surgery and who complained of severe colicky pain. Elective surgery became possible in 92% of patients by gallbladder aspiration. The percentage reached 97 when percutaneous cholecystostomy was added (four patients). Emergent surgery was needed in one patient suffering bile leakage following gallbladder aspiration. Colicky pain was controlled soon after the procedure in most cases. Neither major complications nor mortalities were observed in the following surgical therapies. It is suggested that gallbladder aspiration might be applied as a temporary measure for acute cholecystitis in average-surgical-risk patients, although early surgery should remain the primary choice of therapy in such patients.

Treatment strategy for Boerhaave's syndrome.

Esophageal rupture is a potentially mortal condition. Rapid and correct diagnosis, and urgent surgical treatment with esophagectomy is indicated, but conservative and other surgical treatments have also been reported recently. The treatment strategies for esophageal rupture are discussed here, based on our experiences with four cases during the last 10 years. They were admitted urgently and each was treated by a different method. Three of them underwent emergency operations, one undergoing primary closure of the ruptured esophagus, another received a T-tube insertion from the ruptured site with omental flap, and the third an esophagogastrectomy. The fourth case was treated conservatively. All patients survived and were discharged 36-144 days post treatment. One of them was readmitted for debridement of necrotic rib. In conclusion, the prompt and accurate diagnosis of esophageal rupture is crucial for a subsequent successful treatment. Conservative treatment or operation including esophagectomy will be determined by the severity of the condition.

Evaluation of lateral lymph node dissection with preoperative chemo-radiotherapy for the treatment of advanced middle to lower rectal cancers.

BACKGROUND AND AIMS: This study examined rectal cancers with lateral lymph node (LN) metastases and whether lateral lymph node dissection (LLD) with or without preoperative chemo-radiotherapy (XRT) benefits patients with rectal cancer. PATIENTS AND METHODS: A total of 452 consecutive cases of curatively resected pT2, pT3, and pT4 middle to lower rectal cancers were retrospectively analyzed. Of these, 265 patients underwent curative LLD and 155 XRT. Data were evaluated with respect to the cumulative percentage of survival. RESULTS: Lateral LN metastases were identified in 7.7% of patients. Of the pT3/pT4 extraperitoneal cancer patients 13.5/18.8% had lateral LN metastases. In the treatment of middle rectal cancers and pT2 extraperitoneal cancers LLD either with or without XRT did not improve survival rate. For the treatment of pT3/pT4 extraperitoneal tumors prior to the introduction of total mesorectal excision (TME) in 1994 LLD plus XRT yielded significantly better survival and local control than conventional surgery without LLD or XRT, although LLD alone did not improve either survival or local recurrence rates. Since 1995 TME with or without subsequent LLD has yielded favorable results for the treatment of extraperitoneal tumors. CONCLUSION: For the treatment of middle rectal cancers and pT2 extraperitoneal cancers LLD either with or without XRT does not improve survival rate. For pT3/pT4 extraperitoneal tumors, which are associated with a high incidence of lateral node metastasis, combining treatment modalities such as TME followed by LLD or XRT followed by TME may be considered.

Pelvic exenteration for advanced colorectal cancer with reconstruction of urinary and sphincter functions.

BACKGROUND: Total pelvic exenteration (TPE) for the treatment of advanced colorectal cancer usually involves a double stoma for faecal and urinary excretion, which reduces patient quality of life. In this study, a stomaless reconstruction method for patients normally requiring TPE was evaluated. METHODS: Five patients underwent stomaless TPE. After removal of the tumour with an adequate surgical margin, the urethra was transected at the urogenital diaphragm and the rectum at the anal canal. An ileal neobladder was constructed and coloanal anastomosis was performed. The major omentum was used to construct a septum between the anastomoses. A transgastric ileus tube was used as an intestinal stent to prevent ileus. RESULTS: All patients were alive 12-39 months after operation. Faecal continence was preserved in four patients whose diverting colostomies were closed. All five patients were able to void urine spontaneously, with daytime continence. All but one, in whom cancer recurred, were mobile in the community. CONCLUSION: Stomaless TPE may be considered for locally advanced colorectal cancers that invade the genitourinary organs, provided that neither the anal canal nor the urogenital diaphragm is affected.

Thymidine phosphorylase expression is useful in selecting adjuvant chemotherapy for stage III gastric cancer.

Thymidine phosphorylase (TP) is an enzyme which converts doxifluridine (5'-DFUR) to 5-fluorouracil (5-FU). To assess whether TP expression is useful for selecting adjuvant chemotherapy in advanced gastric cancer, we compared effects of oral 5'-DFUR and 5-FU and assessed correlation between drug efficacy and TP expression level. We examined TP expression in 286 patients. TP expression was assessed with immunohistochemical staining. When we compared prognosis in two chemotherapy groups with high TP expression, better survival was observed in 5'-DFUR than in 5-FU group (p=0.0413). Especially in stage III, patients with high TP had better survival in 5'-DFUR than in 5-FU group.

Role of inorganic polyphosphate in promoting ribosomal protein degradation by the Lon protease in E. coli.

Inorganic polyphosphate (polyP), a polymer of hundreds of phosphate (Pi) residues, accumulates in Escherichia coli in response to stresses, including amino acid starvation. Here we show that the adenosine 5'-triphosphate-dependent protease Lon formed a complex with polyP and degraded most of the ribosomal proteins, including S2, L9, and L13. Purified S2 also bound to polyP and formed a complex with Lon in the presence of polyP. Thus, polyP may promote ribosomal protein degradation by the Lon protease, thereby supplying the amino acids needed to respond to starvation.

Isolation and characterization of the Enterobacter cloacae cheR mutant defective in phosphate taxis.

A chemotaxis-defective mutant of Enterobacter cloacae IFO3320, designated EC1, was isolated after N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis. Computer-assisted capillary assays showed that EC1 failed to show chemotactic responses to peptone and inorganic phosphate (Pi). Cloning and sequence analysis showed that EC1 is a cheR mutant, suggesting that Pi taxis by E. cloacae is dependent on a methyl-accepting chemotaxis protein(s) (MCP). EC1 was further mutagenized with NTG to construct cheR pstS and cheR pstA double mutants. A recombinant plasmid pECT01.2, which contained the E. cloacae cheR gene, restored the ability of these double mutants to show chemotaxis toward peptone but not Pi. These results suggest that the phosphate-specific transport (Pst) system, together with a MCP(s), is required for detecting Pi in E. cloacae.

Use of 5-FU plus hyperbaric oxygen for treating malignant tumors: evaluation of antitumor effect and measurement of 5-FU in individual organs.

PURPOSE: Hyperbaric oxygen (HBO) has been shown to increase tumor radiosensitivity. Several reports indicate that it also increases sensitivity to alkylating agents, but other reports suggest that it may speed angiogenesis and tumor growth. To throw light on these questions, we investigated the effects of HBO and 5-fluorouracil (5-FU), individually and in combination, on Sarcoma 180 implants in mice. METHODS: We administered 5-FU at a dose of 0.75 mg/mouse six times per week and HBO at 2 atm absolute pressure for 90 min six times per week, both 17 times in total. In combination treatment, HBO was administered immediately after 5-FU injection. RESULTS: Over the treatment period, tumor diameter increased 277.8% in the untreated control group, 244.1% in the group receiving HBO monotherapy, 182.7% in the group receiving 5-FU monotherapy, and 138.5% in the group receiving combination therapy. Concomitant HBO increased accumulation of 5-FU in the tumors, liver, and kidneys, but not in the brain, of recipient animals. CONCLUSIONS: Based on the above results, we conclude that concomitant HBO enhances the effects of 5-FU.

Chemotaxis of the nematode Caenorhabditis elegans toward cycloheximide and quinine hydrochloride.

We investigated the chemotaxis of the nematode Caenorhabditis elegans toward cycloheximide, denatonium benzoate, 6-n-propyl-2-thiouracil, and quinine hydrochloride. Interestingly, C. elegans was strongly attracted to cycloheximide, while avoiding quinine hydrochloride. This is the first report thus far to describe the chemotactic responses of C. elegans toward bitter tastants for humans.

Mutational analysis of the multicopy hao gene coding for hydroxylamine oxidoreductase in Nitrosomonas sp. strain ENI-11.

The ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11 contains three copies of the hao gene (hao1, hao2, and hao3) coding for hydroxylamine oxidoreductase (HAO). Three single mutants (hao1::kan, hao2::kan, or hao3::kan) had 68 to 75% of the wild-type growth rate and 58 to 89% of the wild-type HAO activity when grown under the same conditions. A double mutant (hao1::kan and hao3::amp) also had 68% of the wild-type growth and 37% of the wild-type HAO activity.

Involvement of an extracellular protease in algicidal activity of the marine bacterium Pseudoalteromonas sp. strain A28.

The marine bacterium Pseudoalteromonas sp. strain A28 was able to kill the diatom Skeletonema costatum strain NIES-324. The culture supernatant of strain A28 showed potent algicidal activity when it was applied to a paper disk placed on a lawn of S. costatum NIES-324. The condensed supernatant, which was prepared by subjecting the A28 culture supernatant to ultrafiltration with a 10,000-M(w)-cutoff membrane, showed algicidal activity, suggesting that strain A28 produced extracellular substances capable of killing S. costatum cells. The condensed supernatant was then found to have protease and DNase activities. Two Pseudoalteromonas mutants lacking algicidal activity, designated NH1 and NH2, were selected after N-methyl-N'-nitrosoguanidine mutagenesis. The culture supernatants of NH1 and NH2 showed less than 15% of the protease activity detected with the parental strain, A28. The protease was purified to homogeneity from A28 culture supernatants by using ion-exchange chromatography followed by preparative gel electrophoresis. Paper-disk assays revealed that the purified protease had potent algicidal activity. The purified protease had a molecular mass for 50 kDa, and the N-terminal amino acid sequence was determined to be Ala-Thr-Pro-Asn-Asp-Pro. The optimum pH and temperature of the protease were found to be 8.8 and 30 degrees C, respectively, by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate. The protease activity was strongly inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, antipain, chymostatin, and leupeptin. No significant inhibition was detected with EDTA, EGTA, phenanthroline or tetraethylenepentamine. These results suggest that Pseudoalteromonas sp. strain A28 produced an extracellular serine protease which was responsible for the algicidal activity of this marine bacterium.