Clavicle fracture with osteomyelitis after neck dissection and post-operative radiotherapy: case report.

BACKGROUND: Non-traumatic bone fractures in cancer patients are usually pathological fractures due to bone metastases. In head and neck cancer patients, clavicle stress fractures may occur as a result of atrophy of the trapezius muscle after neck dissection in which the accessory nerve becomes structurally or functionally damaged. CASE REPORT: A 71-year-old man underwent modified radical neck dissection with accessory nerve preservation and post-operative radiotherapy for submandibular lymph node metastases of tongue cancer. Four weeks after the radiotherapy, a clavicle fracture, with osteomyelitis and abscess formation in the pectoralis major muscle, occurred. Unlike in simple stress fracture, long-term antibiotic administration and drainage surgery were required to suppress the inflammation. CONCLUSION: As seen in the present patient, clavicle stress fractures may occur even after neck dissection in which the accessory nerve is preserved, and may be complicated by osteomyelitis and abscess formation owing to risk factors such as radiotherapy, tracheostomy and contiguous infection.

New screening methods for probiotics with adhesion properties to sialic acid and sulphate residues in human colonic mucin using the Biacore assay.

AIMS: To determine the relationship between adhesive ability of probiotics and acidic residues in human colonic mucin, we developed a new screening method using Biacore to evaluate adherence of bacteria before and after sialic acid or sulphate residues were blocked or removed from mucin. METHODS AND RESULTS: Ten strains of lactobacilli and three strains of bifidobacteria isolated from human faeces were evaluated for their adhesive properties to soluble human colonic mucin (sHCM) using the Biacore binding assay. Three strains (Lactobacillus strain ME-522, Lact. gasseri ME-527 and Bifidobacterium bifidum MCC1092) showing significant adherence were selected. Decreased binding activities were observed after removing sialic acid of sHCM using sialidase. However, after removing the sulphate residue using sulphatase, the adhesion of ME-527 decreased; whereas the remaining two strains had increased adhesion. The adhesion of three probiotics significantly decreased after the sulphate residue was blocked by elution with barium chloride. CONCLUSIONS: A new evaluation method using the Biacore assay was developed to observe binding properties to the acidic residues of sHCM. Results indicated that there was a strong relationship between probiotic adhesion and acidic residues of sHCM. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report showing a screening method that quantitatively measures the binding between bacteria and acidic residues in sHCM using the Biacore binding assay; and provides a new method for the selection of probiotics in the future.

Expression and translocation of aquaporin-2 in the endolymphatic sac in patients with Meniere's disease.

Meniere's disease, characterised by episodic vertigo, fluctuating hearing loss and tinnitus, can occur under conditions of stress. Its pathology was first revealed to be inner ear hydrops through temporal bone studies in 1938. Although its pathogenesis has been proposed to be a disorder of water transport in the inner ear, subsequently, it remains unsolved, until now. A recent study revealed that both plasma stress hormone, vasopressin (pAVP) and its receptor, V2 (V2R) expression in the inner ear endolymphatic sac were significantly higher in Meniere's patients. In the present study, to link V2R-related molecules and inner ear hydrops, we examined V2R-linked water channel molecule, aquaporin-2 (AQP2) expression and translocation in human endolymphatic sac. AQP2 mRNA expression in the endolymphatic sac was significantly higher in Meniere's patients by using real-time polymerase chain reaction, as further confirmed by western blotting. AQP2-like immunoreactivity (-LIR) was translocated from luminal to basolateral side with endosomal trapping in the endolymphatic sac at the time of AVP exposure in human endolymphatic sac tissue culture. The similar AQP2-LIR translocation was also demonstrated by forskolin and blocked by vasopressin/V2R specific antagonist, OPC31260 and protein kinase A (PKA) specific antagonists, H-89 and KT-5720. We concluded that in the pathogenesis of inner ear hydrops resulting in Meniere's attacks, pAVP elevation as a result of stress and subsequent V2R-cAMP-PKA-AQP2 activation and endosomal trapping of AQP2 in the endolymphatic sac, might be important as a basis of this disease. Further experimental and clinical studies are needed to better clarify the neuroscientific relationship between stress and Meniere's disease.

Identification of a new adhesin-like protein from Lactobacillus mucosae ME-340 with specific affinity to the human blood group A and B antigens.

AIMS: To identify and characterize a new adhesin-like protein of probiotics that show specific adhesion to human blood group A and B antigens. METHODS AND RESULTS: Using the BIACORE assay, the adhesion of cell surface components obtained from four lactobacilli strains that adhered to blood group A and B antigens was tested. Their components showed a significant adhesion to A and B antigens when compared to the bovine serum albumin (BSA) control. The 1 mol l(-1) GHCl fraction extracted from Lactobacillus mucosae ME-340 contained a 29-kDa band (Lam29) using SDS-PAGE. The N-terminal amino acid sequence and homology analysis showed that Lam29 was 90% similar to the substrate-binding protein of the ATP-binding cassette (ABC) transporter from Lactobacillus fermentum IFO 3956. The complete nucleotide sequence (858 bp) of Lam29 was determined and encoded a protein of 285 amino acid residues. Phylogenetic analysis and multiple sequence alignments indicated this protein may be related to the cysteine-binding transporter. CONCLUSIONS: The adhesion of ME-340 strain to blood group A and B antigens was mediated by Lam29 that is a putative component of ABC transporter as an adhesin-like protein. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus mucosae ME-340 expressing Lam29 may be useful for competitive exclusion of pathogens via blood group antigen receptors in the human gastrointestinal mucosa and in the development of new probiotic foods.

Behavioral assessment and identification of a molecular marker in a salicylate-induced tinnitus in rats.

Tinnitus is a non-observable phantom sensation. As such, it is a difficult condition to investigate and, to date, no effective treatment has been developed. To approach this phantom sensation, we aimed to develop a rat behavioral model of tinnitus using salicylate, an active component of aspirin known to induce tinnitus. We also aimed to establish a molecular marker of tinnitus by assessing the expression of transient receptor potential cation channel superfamily V-1 (TRPV1) in the rat auditory pathway during salicylate-induced tinnitus. Animals were trained to perform "an active avoidance task": animals were conditioned by electrical footshock to move to the other side of the conditioning box when hearing a sound. Animals received a single injection of saline or salicylate (400 mg/kg i.p.) and false positive responses were measured 2 h after injection as the number of movements during a silent period. The number of responses in salicylate-treated animals was highest when the conditioned stimulus was 60 dB sound pressure level (SPL) and 16 kHz. This indicates that animals could feel tinnitus 2 h after salicylate injection, equivalent to that induced by 60 dB SPL and 16 kHz. By means of real-time PCR and western blot analysis, TRPV1 expression was significantly upregulated in spiral ganglion cells 2 h after salicylate injection and this upregulation together with the increase in the number of false positive responses was significantly suppressed by capsazepine (10 mg/kg i.p.), a specific antagonist of TRPV1. This suggests that salicylate could induce tinnitus through activation of TRPV1 in the rat auditory pathway.

A rare case of a glass foreign body in the parapharyngeal space: pre-operative assessment by contrast-enhanced CT and three-dimensional CT images.

The parapharyngeal space is an infrequent area for foreign bodies to lodge. However, the presence of trauma or inflammation near or within the space is dangerous because of its anatomical proximity to the bifurcation of the maxillary artery, carotid artery and jugular vein. We encountered a rare case, when a glass flask burst, in which intraparotid damage to the facial nerve was seen, as well as a glass foreign body lodged in the parapharyngeal space close to the above named great vessels. We emphasise the usefulness of contrast-enhanced CT and three-dimensional CT images for pre-operative evaluation of the locational relationship between the foreign body and great vessels in the parapharyngeal space.

Transcriptional silencing of ETS-1 efficiently suppresses angiogenesis of pancreatic cancer.

In this study, we addressed the hypothesis that transcriptional suppression of erythroblastosis virus E26 oncogene homolog 1 (ETS-1) is an efficient therapeutic approach to pancreatic adenocarcinoma by investigating the effect of ETS-1 suppression in human pancreatic cancer cells. We accomplished this by using an adenoviral vector encoding only the DNA-binding domain of wild-type ETS-1 (ETS-1 dominant negative, ETS-1-DN). ETS-1-DN decreases ETS-1-binding by competing for its binding to DNA. Adenoviral-mediated transfer of ETS-1-DN (adenoviral ETS-1-DN construct, AdETS-1-DN) into pancreatic tumor cell lines did not affect their proliferation rate in vitro but did significantly inhibit their in vivo growth in nude mice. Furthermore, to test the efficacy of ETS-1-DN in vivo, we injected the AdETS-1-DN into established human pancreatic adenocarcinomas grown in nude mice. This treatment significantly reduced tumor size as compared to saline injection, without any detectable side effects. Microvessel density in mouse xenografts displayed significantly lower values in tumors in which ETS-1 was downregulated. In addition, expression of the ETS-1-DN in the pancreatic cancer cells resulted in downregulation of urokinase-type plasminogen activator (u-PA) and metalloproteinase-1 (MMP-1) expression. Taken together, these data suggest that transcriptional inactivation of ETS-1 is able to significantly affect angiogenesis and growth of pancreatic cancer. This effect may be due in part to downregulation of MMP-1 and u-PA expression. Our results suggest that ETS-1-DN is a promising candidate for antiangiogenic gene therapy in pancreatic cancer.

Meniere's attacks occur in the inner ear with excessive vasopressin type-2 receptors.

Meniere's disease is peculiar to humans and is characterised by episodic vertigo, fluctuating hearing loss and tinnitus, and attacks of the affliction occurring under conditions of stress. Its pathology was first revealed to be inner ear hydrops through temporal bone studies in 1938. Although subsequently proposed as a disorder of water metabolism in the inner ear, its pathogenesis remains unsolved. The present study aimed to assess the link between the inner ear pathology in Meniere's disease and vasopressin, an anti-diuretic stress hormone with a potential role in inner ear fluid homeostasis. Blood samples were obtained from Meniere's disease patients in the morning, before any surgical treatment, to examine plasma vasopressin (pAVP) levels, and then from inner ear tissue during surgical treatment, to examine vasopressin type-2 receptor (V2R) in the endolymphatic sac. pAVP and the relative V2R mRNA expression in the endolymphatic sac were examined using a real-time polymerase chain reaction. Relative cAMP activity in the endolymphatic sac was also examined using tissue culture and cAMP assay. Both pAVP (1.6-fold versus controls; P = 0.048) and inner ear V2R mRNA expression (41.5-fold versus controls; P = 0.022) were significantly higher in Meniere's patients. cAMP activity was basally up-regulated (2.1-fold versus controls) and cAMP sensitivity to vasopressin application was largely elevated (4.9-fold versus controls) in Meniere's patients. We conclude that, in the pathogenesis of inner ear hydrops, resulting in Meniere's attacks, elevation of pAVP levels (probably as a result of stress) may present as a matter of consequence, but susceptibility of the V2R-overexpressed and cAMP-hypersensitized inner ear to pAVP elevation might be essential as the basis of this disease. Further experimental and clinical studies are needed to better clarify the relationship between Meniere's disease and stress.

Superficial cooling inhibits force loss in damaged muscle.

The purpose of the study was to determine the effect of muscle damage with and without superficial cooling on force and neural activation of the triceps surae muscles. Seven men performed maximal plantarflexion contractions with and without superficial cooling over the medial gastrocnemius, before, immediate after, and 2 days after transcutaneous electrical stimulation for the medial gastrocnemius, respectively. Transcutaneous electrical stimulation was used to induce muscle damage. The normalized value, which was expressed as percentages of the corresponding relative values obtained before transcutaneous electrical stimulation to after transcutaneous electrical stimulation, of peak torque and integrated electromyogram for the soleus were significantly greater with than without superficial cooling. There was a significant correlation in normalized integrated electromyogram between the medial gastrocnemius and soleus. We conclude that superficial cooling appears to have reduced the magnitude of force loss during maximal voluntary contraction following damage to one of the muscles and the synergistic muscle activation may have contributed to the better force maintenance. The implications of this study are that TES enables more selective damage of muscle than standard protocols, and that superficial cooling over a damaged muscle may have an important role in the acute treatment of muscle injuries.

Cell surface Lactobacillus plantarum LA 318 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) adheres to human colonic mucin.

AIMS: To characterize the adhesion molecule of Lactobacillus plantarum LA 318 that shows high adhesion to human colonic mucin (HCM). METHODS AND RESULTS: The adhesion test used the BIACORE assay where PBS-washed bacterial cells showed a significant decrease in adherence to HCM than distilled water-washed cells. A component in the PBS wash fraction adhered to the HCM and a main protein was detected as a c. 40-kDa band using SDS-PAGE. Using homology comparisons of the N-terminal amino acid sequences compared with sequence databases, this protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The DNA sequence of LA 318 GAPDH was 100% identical to the GAPDH (gapB) of L. plantarum WCFS1. The purified GAPDH adhered to HCM. CONCLUSIONS: We found the adhesin of L. plantarum LA 318 to HCM in its culture PBS wash fraction. The molecule was identified as GAPDH. Because LA 318 possesses the same adhesin as many pathogens, the lactobacilli GAPDH may compete with pathogens infecting the intestine. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report showing GAPDH expressed on the cell surface of lactobacilli adheres to mucin suggesting L. plantarum LA 318 adheres to HCM using GAPDH binding activity to colonize the human intestinal mucosa.

Quantitative evaluation of adhesion of lactobacilli isolated from human intestinal tissues to human colonic mucin using surface plasmon resonance (BIACORE assay).

AIMS: To isolate lactobacilli from the mucus layer of the human intestine and evaluate their adhesion abilities using a BIACORE assay. METHODS AND RESULTS: Thirty strains of lactobacilli were isolated from the mucus layer of normal human intestinal tissues using conventional plate culture. The strains were identified using homology comparisons of the 16S rDNA sequence to databases as Lactobacillus salivarius (26%), Lactobacillus fermentum (13%), Lactobacillus gasseri (10%), Lactobacillus paracasei (7%), Lactobacillus casei (3%), Lactobacillus mucosae (3%) and Lactobacillus plantarum (3%). Lactobacillus plantarum LA 318 shows the highest adhesion to human colonic mucin (HCM) using the BIACORE assay at 115.30 +/- 12.37 resonance unit (RU). The adhesion of cell wall surface proteins from strain LA 318 was significantly higher to HCM than to bovine serum albumin (BSA; P < 0.05). CONCLUSIONS: We isolated 30 strains of lactobacilli. Lactobacillus salivarius was the predominant species of lactobacilli isolated in this study. The adhesion of strain LA 318 isolated from human transverse colon to its mucin was shown. The adhesion could be mediated by lectin-like components on the bacterial cell surface. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study where lactobacilli were isolated from human intestinal tissues and shown to adhere to HCM.

Enhancement of seed vigour following insecticide and phenolic elicitor treatment.

Thiamethoxam (CGA 293'343) is a novel broad-spectrum neonicotinoid insecticide. It is commercially used as a seed treatment under the trademark Cruiser (CRZ). Although many reports detail its insecticidal, plant-protecting properties, there are minimal reports concerning the effect on seed germination activities which can be key control points of seedling vigour. In this report, we investigated the effect of CRZ, fish protein hydrolysates (FPH; a known elicitor of pentose-phosphate pathway) and the combination of CRZ and FPH (CF) on seed vigour of pea, soybean and corn. Seed vigour was investigated by estimating germination percentage, shoot height, shoot weight, total soluble phenolic content, antioxidant content, G6PDH (glucose-6-phosphate dehydrogenase) activity, and GPX (guaiacol peroxidase) activity. Addition of FPH to CRZ (CF) seemed to have a slightly positive effect on seed vigour, especially, CF and FPH treatment for corn and FPH treatment for pea, suggesting that pre-sowing treatments may cause positive/negative effects on seed vigour, depending on the concentration of treatments. Further research will be needed to determine their effects and the optimal concentration for seed priming.

Effects of vascular occlusion on maximal force, exercise-induced T2 changes, and EMG activities of quadriceps femoris muscles.

The purpose of our study was to determine the effect of vascular occlusion on neuromuscular activation and/or the energy metabolic characteristics of the quadriceps femoris (QF) muscles during muscle contractions. Seven men participated in the study. An occlusion cuff was attached to the proximal end of the right thigh, so that blood flow in the anterior medial malleolar artery was reduced to approximately 88 % of the non-occluded flow. Muscle functional magnetic resonance imaging and maximal voluntary contraction (MVC) were carried out before and immediately after 5 sets of 10 repetitions of knee extension exercises at 50 % of the 10 repetitions maximum, from which transverse relaxation times (T2) and maximal force were measured, respectively. Integrated electromyography (iEMG) activity was recorded from the belly of the rectus femoris, vastus lateralis, and vastus medialis muscles during MVC and repetitive exercises. The percentage change in T2 was significantly increased for individual QF muscles, and there was a significant increase in iEMG activity over the 5 sets of repetitive exercises under conditions of vascular occlusion, but there was no significant effect on isometric force and iEMG activity during MVC. These results are consistent with the idea that there is greater osmolite accumulation during exercise with occlusion, although increased neural activation cannot be ruled out.

AURKA is one of the downstream targets of MAPK1/ERK2 in pancreatic cancer.

DUSP6/MKP-3, a specific inhibitor of MAPK1/ERK2, frequently loses its expression in primary pancreatic cancer tissues. This evidence suggests that constitutive activation of MAPK1 synergistically induced by frequent mutation of KRAS2 and the loss of function of DUSP6 plays key roles in pancreatic carcinogenesis and progression. By profiling of gene expressions associated with downregulation of MAPK1 induced by exogenous overexpression of DUSP6 in pancreatic cancer cells, we found that AURKA/STK15, the gene encoding Aurora-A kinase, which plays key roles in cellular mitosis, was among the downregulated genes along with its related genes, which included AURKB, TPX2 and CENPA. An association of expression and promoter activity of AURKA with MAPK activity was verified. Knockdown of ETS2 resulted in a reduction of AURKA expression. These results indicate that AURKA is a direct target of the MAPK pathway and that its overexpression in pancreatic cancer is induced by hyperactivation of the pathway, at least via ETS2.

Genome-wide profiling of promoter methylation in human.

DNA methylation in the promoter region of a gene is associated with a loss of that gene's expression and plays an important role in gene silencing. The inactivation of tumor-suppressor genes by aberrant methylation in the promoter region is well recognized in carcinogenesis. However, there has been little study in this area when it comes to genome-wide profiling of the promoter methylation. Here, we developed a genome-wide profiling method called Microarray-based Integrated Analysis of Methylation by Isoschizomers to analyse the DNA methylation of promoter regions of 8091 human genes. With this method, resistance to both the methylation-sensitive restriction enzyme HpaII and the methylation-insensitive isoschizomer MspI was compared between samples by using a microarray with promoter regions of the 8091 genes. The reliability of the difference in HpaII resistance was judged using the difference in MspI resistance. We demonstrated the utility of this method by finding epigenetic mutations in cancer. Aberrant hypermethylation is known to inactivate tumour suppressor genes. Using this method, we found that frequency of the aberrant promoter hypermethylation in cancer is higher than previously hypothesized. Aberrant hypomethylation is known to induce activation of oncogenes in cancer. Genome-wide analysis of hypomethylated promoter sequences in cancer demonstrated low CG/GC ratio of these sequences, suggesting that CpG-poor genes are sensitive to demethylation activity in cancer.

The effects of ice application over the vastus medialis on the activity of quadriceps femoris assessed by muscle function magnetic resonance imaging.

AIM: The purpose of this study was to investigate the effect of ice application for the muscle vastus medialis (VM) on the activation pattern of the quadriceps femoris muscle during repetitive knee extensions using muscle function magnetic resonance imaging (mfMRI) technique. METHODS: Seven men underwent transverse relaxation time (T2)-weighted magnetic resonance imaging (spin echo, TR/TE=1500/25, 80 ms, 10 mm slice thickness and gap) of their right thigh at rest and immediately after isotonic knee extension exercise with 5 sets of 10 repetitions at a load equal to 70% of their 10 repetitions with and without skin cooling. Cooling over surface skin of the VM was carried out for 3 min before and during 60-s of each rest interval between the knee extension exercise. RESULTS: The relative change in T2 of the muscle vastus intermedius increased significantly more by skin cooling than the control (p<0.01), but not the muscle rectus femoris (RF), muscle vastus lateralis, and VM. CONCLUSION: These results suggest that selective skin cooling combined with repeated muscle contraction facilitates the activation of other synergistic muscles, making this technique useful for activating the agonist muscles expected for injured muscle in training and rehabilitation.

Vestibular function and vasopressin.

OBJECTIVE: To investigate the relation between the vestibular system and vasopressin (AVP). MATERIAL AND METHODS: We examined the effects of electrical and caloric vestibular stimulation on plasma AVP levels in anesthetized rats. Plasma AVP levels of patients with intractable Ménière's disease who were subjected to endolymphatic drainage and steroid instillation surgery (EDSS) or intratympanic gentamicin (GM) injection were measured before and after these interventions. RESULTS: Electrical vestibular stimulation increased plasma AVP levels in a current intensity-dependent manner. Plasma AVP levels were also increased by caloric stimulation with cold water. Plasma AVP levels decreased rapidly after EDSS, and were maintained at a low level even 6-12 months following EDSS or intratympanic GM injection. CONCLUSIONS: Vestibular activation or inhibition-induced imbalance of intervestibular activities increased plasma AVP levels in rats. Therefore, vestibular disorder would seem to increase plasma AVP and thus worsen endolymphatic hydrops. EDSS rapidly decreased plasma AVP and would seem to reduce hydrops. Inhibition of vertigo spells by EDSS or intratympanic GM injection would reduce a possible stress response, resulting in a decrease in plasma AVP levels a long time after the treatments. This resultant decrease in AVP would beneficially inhibit the formation and/or maintenance of hydrops and thus prevent vertigo spells.

Overexpression of the p53-inducible brain-specific angiogenesis inhibitor 1 suppresses efficiently tumour angiogenesis.

The brain-specific angiogenesis inhibitor 1 gene has been isolated in an attempt to find fragments with p53 "functional" binding sites. As reported herein and by others, brain-specific angiogenesis inhibitor 1 expression is present in some normal tissues, but is reduced or lost in tumour tissues. Such data and its particular structure prompted the hypothesis that brain-specific angiogenesis inhibitor 1 may act as a mediator in the local angiogenesis balance. We herein demonstrate that brain-specific angiogenesis inhibitor 1 over-expression suppresses tumour angiogenesis, delaying significantly the human tumour growth in immunodeficient mice. The inhibitory effect of brain-specific angiogenesis inhibitor 1 was documented using our intravital microscopy system, strongly implicating brain-specific angiogenesis inhibitor 1 as a mediator in the control of tumour angiogenesis. In contrast, in vitro tumour cell proliferation was not inhibited by brain-specific angiogenesis inhibitor 1 transfection, whereas some level of cytotoxicity was assessed for endothelial cells. Immunohistochemical analysis of tumour samples confirmed a reduction in the microvessel density index in brain-specific angiogenesis inhibitor 1-overexpressing tumours. At messenger level, moderate changes could be detected, involving the down-regulation of vascular endothelial growth factor and collagenase-1 expression. Furthermore, brain-specific angiogenesis inhibitor 1 expression that was lost in a selection of human cancer cell lines could be restored by wild-type p53 adenoviral transfection. Brain-specific angiogenesis inhibitor 1 should be considered for gene therapy and development of efficient drugs based on endogenous antiangiogenic molecules.

Human BAC contig covering the deleted region in pancreatic cancer at 12q21.

In sporadic human primary pancreatic cancer tissues, loss of heterozygosity is frequently observed in the 1-cM region between D12S81 and D12S1719 at 12q21. Loss of this chromosome arm is known to be associate with a poor prognosis in pancreatic cancer patients. Herein we report a complete contig of human bacterial artificial chromosome (BAC) clones covering the deleted region. The region was covered by 21 BAC clones in a minimum tiling path. The clones were confirmed to exist at 12q21 by fluorescence in situ hybridization. We identified novel 40 sequence tagged sites and mapped 10 expressed sequence tags in this region. The BAC contig reported here provides an avenue for determining the complete nucleotide sequence and mining putative tumor suppressor genes in the deleted region of pancreatic cancer at 12q21.