Alkaline Stress Causes Changes in Polyamine Biosynthesis in Thermus thermophilus.

An extreme thermophile, Thermus thermophilus, produces 16 different polyamines including long-chain and branched-chain polyamines. The composition and content of polyamines in the thermophile cells change not only with growth temperature but also with pH changes. In particular, cell growth decreased greatly at alkaline medium together with significant changes in the composition and content of polyamines. The amounts of tetraamines (spermine and its homologs) markedly decreased at alkaline pH. Thus, we knocked out the speE gene, which is involved in the biosynthesis of tetraamines, and changes of composition of polyamines with pH changes in the mutant cells were studied. Cell growth in the ΔspeE strain was decreased compared with that of the wild-type strain for all pHs, suggesting that tetraamines are important for cell proliferation. Interestingly, the amount of spermidine decreased and that of putrescine increased in wild-type cells at elevated pH, although T. thermophilus lacks a putrescine synthesizing pathway. In addition, polyamines possessing a diaminobutane moiety, such as spermine, decreased greatly at high pH. We assessed whether the speB gene encoding aminopropylagmatine ureohydrolase (TtSpeB) is directly involved in the synthesis of putrescine. The catalytic assay of the purified enzyme indicated that TtSpeB accepts agmatine as its substrate and produces putrescine due to the change in substrate specificity at high pH. These results suggest that pH stress was exacerbated upon intracellular depletion of polyamines possessing a diaminobutane moiety induced by unusual changes in polyamine biosynthesis under high pH conditions.

[A Case of Postoperative Pulmonary Metastasis of Breast Cancer with Complete Response by Abemaciclib plus Fulvestrant Therapy].

A 66-year-old woman underwent total mastectomy with level Ⅰ and Ⅱ axillary lymph node dissection for right breast cancer in July 2007. The pathology results indicated the presence of T2N0M0 invasive ductal carcinoma(tubule forming type), that was estrogen receptor-positive and human epidermal growth factor 2-negative. She received postoperative adjuvant therapy with oral anastrozole(ANA)for 5 years. Eleven years after surgery, at the age of 77 years, a chest X-ray examination during a routine health checkup identified a mass shadow in the right lung. Further investigation revealed bilateral multiple lung metastases due to breast cancer recurrence. Histological examination of a tissue obtained by computed tomography(CT)-guided lung biopsy confirmed that the histological type and subtype were identical to those found in the initial surgery. Hence, endocrine therapy with ANA plus CDK4/6 inhibitor was started in November 2018. However, the first CDK4/6 inhibitor, palbociclib, caused severe myelosuppression even when the dose was reduced by 2 levels. Therefore in January 2019, the patient was switched to abemaciclib, with the dose reduced by 1 level initially and then reduced by 2 levels from August 2019. In June 2019, new multiple lung metastases appeared, and the patient was switched from ANA to fulvestrant, after which complete response was achieved in 6 months. CT in June 2021 showed no recurrence, and the patient(now 80-year-old)continues to take abemaciclib plus fulvestrant therapy.

[Primary Peritoneal Carcinoma with Long Term Survival-A Case Report].

A 62-year-old woman underwent upper endoscopy in January 2009 to reveal the presence of an extrinsic compression measuring approximately 3 cm in the anterior wall of the gastric antrum. Further examinations suggested that it was caused by peritoneal cancer of an unknown origin; thus, staging laparoscopy was performed in May 2009. Multiple white nodules of varying sizes were found scattered throughout the right upper quadrant of the abdomen and the right abdomen. Based on a biopsy of the greater omentum, the patient was diagnosed with papillary serous adenocarcinoma. As no abnormalities were observed in the uterus and ovary, it was suspected that the patient had primary peritoneal cancer. Hence, in July 2009, the patient underwent resection of the greater omentum, gastric pylorus, gall bladder, and right hemicolon where the tumors were localized, as well as bilateral adnexectomy. Based on intraoperative findings and postoperative histology, the patient was diagnosed with high-grade primary peritoneal serous adenocarcinoma and received paclitaxel and carboplatin therapy. Subsequent follow-up examinations, including positron emission tomography-computed tomography(PET-CT), indicated repeated recurrences in the mesentery, the pelvic floor, and around the remnant stomach. After identifying these recurrences, the patient was treated with platinum-based drugs, experiencing repeated response and cessation cycles. Since September 2019, the patient has received olaparib therapy. PET-CT examination performed in September 2020 indicated that the patient remained in complete remission.

Long and branched polyamines are required for maintenance of the ribosome, tRNAHis and tRNATyr in Thermus thermophilus cells at high temperatures.

Thermus thermophilus is an extremely thermophilic eubacterium that produces various polyamines. Aminopropylagmatine ureohydrolase (SpeB) and SAM decarboxylase-like protein 1 (SpeD1) are involved in the biosynthesis of spermidine from arginine. Because long and branched polyamines in T. thermophilus are synthesized from spermidine, the speB and speD1 gene-deleted strains (ΔspeB and ΔspeD1, respectively) cannot synthesize long and branched polyamines. Although neither strain grew at high temperatures (>75°C) in minimal medium, both strains survived at 80°C when they were cultured at 70°C until the mid-log phase and then shifted to 80°C. We therefore prepared the ΔspeB and ΔspeD1 cells using this culture method. Microscopic analysis showed that both strains can survive for 10 h after the temperature shift. Although the modification levels of 2'-O-methylguanosine at position 18, N7 -methylguanosine at position 46, 5-methyluridine at position 54 and N1 -methyladenosine at position 58 in the class I tRNA from both strains were normal, amounts of tRNATyr , tRNAHis , rRNAs and 70S ribosomes were decreased after the temperature shift. Furthermore, in vivo protein synthesis in both strains was completely lost 10 h after the temperature shift. Thus, long and branched polyamines are required for at least the maintenance of 70S ribosome and some tRNA species at high temperatures.

[A Case of Recurrent Breast Cancer with Complete Response to Eribulin Monotherapy after Bevacizumab plus Paclitaxel Combination Therapy].

A 65-year-old woman underwent mastectomy and dissection of a level I axillary lymph node in January 2002 for left breast cancer. The diagnosis was T1N0M0 scirrhous carcinoma that was estrogen receptor-positive, progesterone receptorpositive, and human epidermal growth factor receptor 2-negative. After 3 years 10 months, during which the patient underwent adjuvant therapy with oral aromatase inhibitors, she developed bilateral multiple lung metastases. These were treated with the anticancer agents anthracycline and taxane. Progressive disease(more and larger lung metastases)was diagnosed in April 2013, and bevacizumab plus paclitaxel combination therapy was started. After completion of 4 courses, a lung abscess appeared, which was conjectured to represent rapid tumor necrosis that had become infected. As several tumors remained solid even after the lung abscess improved, the patient received 18 courses of eribulin monotherapy. Computed tomography in April 2016 revealed only patches of linear or cord-like scarring in both lungs, with no metastatic or recurrent foci. In this case, a patient with recurrent breast cancer responded to the sequential administration of bevacizumab plus paclitaxel combination therapy followed by eribulin monotherapy.

[Total Gastrectomy for Advanced Gastric Cancer after Coronary Artery Bypass Grafting Using the Right Gastroepiploic Artery: Report of a Case].

An 82-year-old man, who had undergone coronary artery bypass grafting(CABG) with the right gastroepiploic artery( RGEA) 13 years previously, suffered with abdominal pain after meal and tarry stools, and was diagnosed with advanced gastric cancer. Gastroscopy revealed an advanced Borrmann type 4 cancer at the lesser curvature of the gastric body to the pyloric ring. The gastrogram showed poor extension and stenosis at the same part. Abdominal computed tomography showed the tumor reached the subserosal layer and infrapyloric lymph nodes were swollen. Abdominal angiography showed the RGEA graft remained well patent. Total gastrectomy with D2 lymph nodes dissection and arterial reconstruction between the splenic artery and the RGEA graft was performed. He has been well without any sign of cancer recurrence since the operation. Recently, more patients with CABG using RGEA are found to have gastric cancer and require the resection of RGEA for lymph nodes dissection as this case. We consider this procedure one of the options for advanced gastric cancer after coronary bypass grafting using RGEA.

Caldomycin, a new guanidopolyamine produced by a novel agmatine homocoupling enzyme involved in homospermidine biosynthesis.

An extreme thermophilic bacterium, Thermus thermophilus produces more than 20 unusual polyamines, but their biosynthetic pathways, including homospermidine, are not yet fully understood. Two types of homospermidine synthases have been identified in plants and bacteria, which use spermidine and putrescine or two molecules of putrescine as substrates. However, homospermidine synthases with such substrate specificity have not been identified in T. thermophilus. Here we identified a novel agmatine homocoupling enzyme that is involved in homospermidine biosynthesis in T. thermophilus. The reaction mechanism is different from that of a previously described homospermidine synthase, and involves conjugation of two molecules of agmatine, which produces a diamidino derivative of homospermidine (caldomycin) as an immediate precursor of homospermidine. We conclude that there is a homospermidine biosynthetic pathway from agmatine via caldomycin synthase followed by ureohydrolase in T. thermophilus. Furthermore, it is shown that caldomycin is a novel compound existing in nature.

Prediction of pancreatic anastomotic failure after pancreatoduodenectomy: the use of preoperative, quantitative computed tomography to measure remnant pancreatic volume and body composition.

OBJECTIVE: To determine whether remnant pancreatic volume (RPV), subcutaneous/visceral adipose tissue(SAT/VAT) area, and skeletal muscle (SM) area calculated from preoperative computed tomography (CT) can predict the occurrence of pancreatic anastomotic failure (PAF) after pancreatoduodenectomy (PD). BACKGROUND: Increased body mass index, small main pancreatic duct, and soft pancreatic texture are well-established predictors of PAF after PD. The impact on PAF of anthropomorphic measurements, such as RPV and body composition, is unknown. METHODS: In 173 patients undergoing PD from 2004 to 2009, cross sections of SAT/VAT/SM area were quantitated volumetrically, respectively, from preoperative CT. RPV was calculated from the CT as the sum of pancreatic tissue area to the left of the presumed pancreatic transection site. The predictive ability for multiple models using combinations of body mass index, RPV, SAT/VAT area, SM area, main pancreatic duct size, and pancreatic gland texture was described using a concordance index (c-index). RESULTS: Clinically relevant PAF occurred in 22 patients (13%). Multivariate logistic regression analysis identified RPV (P = 0.0012), VAT area (P = 0.0003), and SM area (P = 0.0006) as independent predictors of PAF. Using previously identified risk factors, the best 2-predictor model (body mass index and pancreatic duct size) resulted in a c-index of 0.748. Using anthropomorphic factors, however, the 2-predictor model using VAT and SM areas revealed a superior c-index of 0.959. CONCLUSIONS: Our 2-predictor model using VAT area and SM area based on volumetric quantification using preoperative CT may offer clinical benefit as an objective prognostic measure to predict clinically relevant PAF after PD.

Thermus thermophilus polyploid cells directly imaged by X-ray laser diffraction.

Thermus thermophilus is reportedly polyploid and carries four to five identical genome copies per cell, based on molecular biological experiments. To directly detect polyploidy in this bacterium, we performed live cell imaging by X-ray free-electron laser (XFEL) diffraction and observed its internal structures. The use of femtosecond XFEL pulses enables snapshots of live, undamaged cells. For successful XFEL imaging, we developed a bacterial culture method using a starch- and casein-rich medium that produces a predominance of rod-shaped cells shorter than the focused XFEL beam size, which is slightly smaller than 2 µm. When cultured in the developed medium, the length of T. thermophilus cells, which is typically ~4 µm, was less than half its usual length. We placed living cells in a micro-liquid enclosure array and successively exposed each enclosure to a single XFEL pulse. A cell image was successfully obtained by the coherent diffractive imaging technique with iterative phase retrieval calculations. The reconstructed cell image revealed five peaks, which are most likely to be nucleoids, arranged in a row in the polyploid cell without gaps. This study demonstrates that XFELs offer a novel approach for visualizing the internal nanostructures of living, micrometer-sized, polyploid bacterial cells.

Putrescine Biosynthesis from Agmatine by Arginase (TtARG) in Thermus thermophilus.

In the three domains of life, three biosynthetic pathways are known for putrescine. The first route is conversion of ornithine to putrescine by ornithine decarboxylase (ODC: SpeC), the second route is the conversion of arginine to agmatine by arginine decarboxylase (ADC: SpeA), followed by the conversion of agmatine to putrescine by agmatine ureohydrolase (AUH: SpeB), and the third route is the conversion of agmatine to N-carbamoylputrescine by agmatine deiminase (agmatine iminohydrolase, AIH), followed by the conversion of N-carbamoylputrescine to putrescine by N-carbamoylputrescine amidohydrolase (NCPAH). An extreme thermophile, Thermus thermophilus produces putrescine, although this bacterium lacks homologs for putrescine synthesizing pathways, such as ODC, AUH, AIH and NCPAH. To identify genes involved in putrescine biosynthesis in T. thermophilus, putrescine biosynthesis was examined by disruption of a predicted gene for agmatinase (agmatine ureohydrolase), or by using purified enzyme. It was found that arginase (TTHA1496) showed an agmatinase activity utilizing agmatine as a substrate. These results indicate that this bacterium can use arginase for putrescine biosynthesis. Arginase is a major contributor to putrescine biosynthesis under physiological conditions. The presence of an alternative pathway for converting agmatine into putrescine is functionally important for polyamine metabolism supporting survival at extreme environments.

Intraductal papillary mucinous neoplasms of the pancreas: making a disposition using the natural history.

The process of Intraductal papillary mucinous neoplasms (IPMN) follows the adenoma-to-carcinoma sequence. If it progresses to malignancy about 5 years is required. Even though the process is slow IPMN provides the clinician with the opportunity to avoid malignancy if the patient is at risk. The natural history as observed through Kaplan Meier event curves for occurrence of malignancy show the process to malignancy is much faster (50% within 2 years) if pancreatitis-like symptoms are present or if the main pancreatic duct (MPD) is involved. Almost all decisions to resect (95% in our experience) are based on the presence of symptoms or the MPD location. Cyst size is used infrequently. Every patient with an IPMN should always have a planned follow-up and the frequency depends on the perceived risk of malignancy-immediate imaging if becomes symptomatic to every 2 to 3 years if asymptomatic side branch lesions. The natural history provides modern guidelines for making decisions in patients with a newly discovered IPMN.

Postoperative hematological changes after spleen-preserving distal pancreatectomy with preservation of the splenic artery and vein.

BACKGROUND: The aim of this study was to determine the early postoperative hematological changes after spleen-preserving distal pancreatectomy (SpDP) with preservation of the splenic artery and vein (PSAV). METHODS: We reviewed 53 patients who underwent SpDP with PSAV (n = 21) or distal pancreatectomy with splenectomy (DPS; n = 32) for benign or low-grade malignant lesions between July 1998 and June 2010. Red and white blood cell (WBC) count, platelet count, serum hemoglobin, hematocrit, C-reactive protein, albumin level, and clinical factors were compared between the SpDP with PSAV and DPS. RESULTS: There were no significant differences in the patient characteristics between the two groups. Platelet count on postoperative day (POD) 5 and WBC count on POD 3 were significantly higher in the DPS group, and these differences continued to be significant until the 3rd month after surgery. Serum hemoglobin and hematocrit in the 1st month after surgery were also significantly higher in the SpDP with PSAV group. CONCLUSION: The hematological benefits of SpDP with PSAV include reduction of postoperative hematological abnormalities in the early postoperative phase and recovery of the serum hemoglobin and hematocrit levels in the early postoperative phase.

Identification of a novel d-amino acid aminotransferase involved in d-glutamate biosynthetic pathways in the hyperthermophile Thermotoga maritima.

The hyperthermophilic bacterium Thermotoga maritima has an atypical peptidoglycan that contains d-lysine alongside the usual d-alanine and d-glutamate. We previously identified a lysine racemase involved in d-lysine biosynthesis, and this enzyme also possesses alanine racemase activity. However, T. maritima has neither alanine racemase nor glutamate racemase enzymes; hence, the precise biosynthetic pathways of d-alanine and d-glutamate remain unclear in T. maritima. In the present study, we identified and characterized a novel d-amino acid aminotransferase (TM0831) in T. maritima. TM0831 exhibited aminotransferase activity towards 23 d-amino acids, but did not display activity towards l-amino acids. It displayed high specific activities towards d-homoserine and d-glutamine as amino donors. The most preferred acceptor was 2-oxoglutarate, followed by glyoxylate. Additionally, TM0831 displayed racemase activity towards four amino acids including aspartate and glutamate. Catalytic efficiency (kcat /Km ) for aminotransferase activity was higher than for racemase activity, and pH profiles were distinct between these two activities. To evaluate the functions of TM0831, we constructed a TTHA1643 (encoding glutamate racemase)-deficient Thermus thermophilus strain (∆TTHA1643) and integrated the TM0831 gene into the genome of ∆TTHA1643. The growth of this TM0831-integrated strain was promoted compared with ∆TTHA1643 and was restored to almost the same level as that of the wild-type strain. These results suggest that TM0831 is involved in d-glutamate production. TM0831 is a novel d-amino acid aminotransferase with racemase activity that is involved in the production of d-amino acids in T. maritima.

Polyamines produced by an extreme thermophile are essential for cell growth at high temperature.

An extreme thermophile, Thermus thermophilus grows at an optimum temperature of around 70°C and produces 16 different polyamines including long-chain and branched-chain polyamines. We found that the composition of polyamines in the thermophile cells changes with culture temperature. Long-chain and branched-chain polyamines (unusual polyamines) were increased in the cells grown at high temperature such as 80°C, but they were minor components in the cells grown at relatively lower temperature such as 60°C. The effects of polyamines on cell growth were studied using T. thermophilus HB8 ΔspeA deficient in arginine decarboxylase. Cell growth of this mutant strain was significantly decreased at 70°C. This mutant strain cannot produce polyamines and grows poorly at 75°C. It was also determined whether polyamines are directly involved in protecting DNA from DNA double-strand breaks (DSBs) induced by heat. Polyamines protected DNA against double-strand breaks. Therefore, polyamines play essential roles in cell growth at extremely high temperature through maintaining a functional conformation of DNA against DSBs and depurination.

Calditerricola satsumensis gen. nov., sp. nov. and Calditerricola yamamurae sp. nov., extreme thermophiles isolated from a high-temperature compost.

Two novel thermophilic micro-organisms, designated YMO81(T) and YMO722(T), were isolated from a high-temperature compost (internal temperature > 95 °C). The isolates were able to grow at 80 °C in a nutrient broth and in a synthetic medium. Cells were aerobic, Gram-negative rods (0.3 x 4.0 µm). Spore formation was not observed. Strain YMO81(T) grew at 83 °C and pH 6.9-8.9 and grew optimally at 78 °C and pH 7.5 with 2  % NaCl. For growth in a synthetic minimal medium at 70 °C, the vitamins biotin, folic acid and thiamine and the amino acids glutamine and methionine were essential for growth of both strains; at 80 °C, strain YMO81(T) also required histidine, isoleucine, leucine, lysine, phenylalanine, serine, tryptophan and valine. Cellular fatty acids of the isolates comprised mainly iso-C17:0 and anteiso-C17:0. The DNA G+C contents of strains YMO81(T) and YMO722(T) were 70 and 64 mol%, respectively. When the 16S rRNA gene sequences of the isolates were compared with those of other bacteria, highest similarity was observed with Planifilum yunnanense LA5(T) (90 % 16S rRNA gene sequence similarity). DNA-DNA relatedness between strain YMO722(T) and strain YMO81(T) was 55  %. N4-Aminopropylspermine was identified as a major polyamine, which suggested that the isolates were distinct from other related taxa. On the basis of phylogenetic, phenotypic and chemotaxonomic analyses, we propose a new genus, Calditerricola gen. nov., and two novel species, the type species Calditerricola satsumensis sp. nov., with type strain YMO81(T) (=ATCC BAA-1462(T) =JCM 14719(T) =DSM 45223(T)), and Calditerricola yamamurae sp. nov., with type strain YMO722(T) (=ATCC BAA-1461(T) =JCM 14720(T) =DSM 45224(T)).

Complete Genome Sequences of Thermus thermophilus Strains HB5002 and HB5008, Isolated from Mine Hot Spring in Japan.

We isolated Thermus thermophilus strains HB5002 and HB5008 from Mine Hot Spring in Japan. Whole-genome sequencing revealed that they showed ∼100% average nucleotide identity to each other, ≥98.53% to the T. thermophilus strains originating from the same spot but ≤97.64% to the T. thermophilus strains from geographically different places in Japan.

Complete Genome Sequence of Thermus thermophilus Strain HB5018, Isolated from Mine Hot Spring in Japan.

We isolated Thermus thermophilus strain HB5018 from Mine Hot Spring in Japan, where the type strain HB8 was isolated nearly half a century ago. The complete genome sequence of HB5018 showed 99.1% average nucleotide identity with HB8, suggesting strict species conservation in the habitat over the past 50 years.

Enzymatic properties and physiological function of glutamate racemase from Thermus thermophilus.

d-Amino acids are physiologically important components of peptidoglycan in the bacterial cell wall, maintaining cell structure and aiding adaptation to environmental changes through peptidoglycan remodelling. Therefore, the biosynthesis of d-amino acids is essential for bacteria to adapt to different environmental conditions. The peptidoglycan of the extremely thermophilic bacterium Thermus thermophilus contains d-alanine (d-Ala) and d-glutamate (d-Glu), but its d-amino acid metabolism remains poorly understood. Here, we investigated the enzyme activity and function of the product of the TTHA1643 gene, which is annotated to be a Glu racemase in the T. thermophilus HB8 genome. Among 21 amino acids tested, TTHA1643 showed highly specific activity toward Glu as the substrate. The catalytic efficiency (kcat/Km) of TTHA1643 toward d- and l-Glu was comparable; however, the kcat value was 18-fold higher for l-Glu than for d-Glu. Temperature and pH profiles showed that the racemase activity of TTHA1643 is high under physiological conditions for T. thermophilus growth. To assess physiological relevance, we constructed a TTHA1643-deficient strain (∆TTHA1643) by replacing the TTHA1643 gene with the thermostable hygromycin resistance gene. Growth of the ∆TTHA1643 strain in synthetic medium without d-Glu was clearly diminished relative to wild type, although the TTHA1643 deletion was not lethal, suggesting that alternative d-Glu biosynthetic pathways may exist. The deterioration in growth was restored by adding d-Glu to the culture medium, showing that d-Glu is required for normal growth of T. thermophilus. Collectively, our findings show that TTHA1643 is a Glu racemase and has the physiological function of d-Glu production in T. thermophilus.

Intrahepatic micrometastases around liver metastases from gastric cancer.

BACKGROUND/PURPOSE: We aimed to clarify the association between the presence of micrometastases around liver metastases from gastric cancer and the results of hepatic resection. In addition, we investigated the influence of E-cadherin and matrix metalloproteinase (MMP)-7 expression on the development of micrometastases. METHODS: Micrometastases around liver metastases were examined microscopically in 31 metastatic liver tumor specimens resected from 17 patients who had undergone hepatic resection for liver metastases from gastric cancer. E-cadherin and MMP-7 expression in the primary gastric tumor, the liver metastases, and the micrometastases were examined immunohistochemically. RESULTS: Hepatic micrometastases were present in around 48% of the liver metastases, accounting for 59% of the patients. The tumor recurrence rate in the remnant liver after hepatic resection was significantly higher, and survival significantly poorer, in patients with such micrometastases than in those without. Micrometastases tended to appear around the liver metastases that had reduced E-cadherin expression. Most of the micrometastases in the lymph ducts and sinusoids showed reduced E-cadherin expression. MMP-7 expression was not correlated with the presence of micrometastases. CONCLUSIONS: About half of the hepatic metastases from gastric cancer had seeded off micrometastases, and the presence of these micrometastases was associated with a poorer result of hepatic resection. Reduced E-cadherin expression in metastatic liver tumors may be associated with the development of micrometastases.