The expense for one implantation of a banked bone allograft from a cadaveric donor and the issues affecting current advanced medical treatment in the Japanese orthopaedic field

Demand for banked bone allografts is increasing in Japan; however, there are too few bone banks and the bone bank network is not well-established. One reason for this was lack of funding for banks. Bone banks had to bear all material expenses of banked bone allografts themselves because this was not designated a covered expense. In December 2004, the Japanese government started a new “Advanced Medical Treatment” administration system which allowed an approved institution to charge the expense of authorized advanced medical treatments directly to patients. The treatment named “Cryopreserved allogenic bone and ligamentous tissue retrieved from cadaveric donor” was approved as an advanced medical treatment in March 2007. We present the calculation method and the expense per implantation of a banked bone allograft from a cadaveric donor under this treatment and raise issues which affect this advanced medical treatment and remain to be resolved in the Japanese orthopaedic field.     

Endometrial Cancer as a Familial Tumor: Pathology and Molecular Carcinogenesis (Review)

Some cases of endometrial cancer are associated with a familial tumor and are referred to as hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome). Such tumors are thought to be induced by germline mutation of the DNA mismatch repair (MMR) gene, but many aspects of the pathology of familial endometrial cancer are unclear and no effective screening method has been established. However, the pathology of endometrial cancer with familial tumor has been progressively clarified in recent studies. At present, about 0.5% of all cases of endometrial cancers meet the clinical diagnostic criteria for HNPCC. A recent analysis of the three MMR genes (hMLH1, hMSH2 and hMSH6) revealed germline mutations in 18 of 120 cases (15.0%) of endometrial cancer with familial accumulation of cancer or double cancer, with a frameshift mutation of the hMSH6 gene being the most common. Many cases with mutation did not meet the current clinical diagnostic criteria for HNPCC, indicating that familial endometrial cancer is often not diagnosed as HNPCC. The results suggest that the hMSH6 gene mutation may be important in carcinogenesis in endometrial cancer and germline mutations of the MMR gene may be more prevalent in cases associated with familial accumulation of cancer. An international large-scale muticenter study is required to obtain further information about the pathology of endometrial cancer as a familial tumor.Key Words: HNPCC, Endometrial cancer, DNA mismatch repair gene, hMLH1, hMSH6.     

Neurophysiological study to assess the severity of each site through the motor neuron fiber in entrapment neuropathy

The purpose of this study is to evaluate prospectively the sensitivities of conventional and new electrophysiological techniques and to investigate their relationship with the body mass index (BMI) in a population of patients suspected of having carpal tunnel syndrome (CTS). In this study, 165 hands of 92 consecutive patients (81 female, 11 male) with clinical diagnosis of CTS were compared to reference population of 60 hands of 30 healthy subjects (26 female and 4 male). Extensive sensory and motor nerve conduction studies (NCSs) were performed in the diagnosis of subtle CTS patients. Also, the patients were divided into subgroups and sensitivities were determined according to BMI. The mean BMI was found to be significantly higher in the CTS than in the control group (p < 0.001). The sensitivity of the median sensory nerve latency (mSDL) and median motor distal latency (mMDL) were 75.8% and 68.5%, respectively. The most sensitive parameters of sensory and motor NCSs were the difference between median and ulnar sensory distal latencies to the fourth digit [(D4M-D4U), (77%)] and the median motor terminal latency index [(mTLI), (70.3%)], while the median-to-ulnar sensory action potential amplitude ratio (27%) and the median-thenar to ulnar-hypothenar motor action potential amplitude ratio (15%) were least sensitive tests. Sensory tests were more sensitive than motor NCSs. Combining mSDL with D4M-D4U, and mMDL with mTLI allowed for the detection of abnormalities in 150 (91%) and 132 (80%) hands, respectively. Measurements of all NCSs parameters were abnormal in obese than in non-obese patients when compared to the BMI. The newer nerve conduction techniques and combining different NCSs tests are more sensitive than single conventional NCS test for the diagnosis of suspected CTS. Meanwhile, CTS is associated with increasing BMI.     

Global gene expression profiling in early-stage polycystic kidney disease in the Han:SPRD Cy rat identifies a role for RXR signaling

Han:SPRD Cy is a spontaneous rat model of polycystic kidney disease (PKD) caused by a missense mutation in Pkdr1. Cystogenesis in this model is not clearly understood. In the current study, we performed global gene expression profiling in early-stage PKD cyst development in Cy/Cy kidneys and normal (+/+) kidneys at 3 and 7 days of postnatal age. Expression profiles were determined by microarray analysis, followed by validation with real-time RT-PCR. Genes were selected with over 1.5-fold expression changes compared with age-matched +/+ kidneys for canonical pathway analysis. We found nine pathways in common between 3- and 7-day Cy/Cy kidneys. Three significantly changed pathways were designated "Vitamin D Receptor (VDR)/Retinoid X Receptor (RXR) Activation," "LPS/IL-1-Mediated Inhibition of RXR Function," and "Liver X Receptor (LXR)/RXR Activation." These results suggest that RXR-mediated signaling is significantly altered in developing kidneys with mutated Pkdr1. In gene ontology analysis, the functions of these RXR-related genes were found to be involved in regulating cell proliferation and organ morphogenesis. With real-time RT-PCR analysis, the upregulation of Ptx2, Alox15b, OSP, and PCNA, major markers of cell proliferation associated with the RXR pathway, were confirmed in 3- and 7-day Cy/Cy kidneys compared with 3-day +/+ kidneys. The increased RXR protein was observed in both the nucleus and cytoplasm of cystic epithelial cells in early-stage Cy/Cy kidneys, and the RXR-positive cells were strongly positive for PCNA staining. Taken together, cell proliferation and organ morphogenesis signals transduced by RXR-mediated pathways may have important roles for cystogenesis in early-stage PKD in this Pkdr1-mutated Cy rat.     

Systematic analyses of free ceramide species and ceramide species comprising neutral glycosphingolipids by MALDI-TOF MS with high-energy CID

Free ceramides and glycosphingolipids (GSLs) are important components of the membrane microdomain and play significant roles in cell survival. Recent studies have revealed that both fatty acids and long-chain bases (LCBs) are more diverse than expected, in terms of i) alkyl chain length, ii) hydroxylation and iii) the presence or absence of double bonds. Electrospray ionization mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been well utilized to characterize sphingolipids with high throughput, but reports to date have not fully characterized various types of ceramide species such as hydroxyl fatty acids and/or trihydroxy-LCBs of both free ceramides and the constituent ceramides in neutral GSLs. We performed a systematic analysis of both ceramide species, including LCBs with nona-octadeca lengths using MALDI-TOF MS with high-energy collision-induced dissociation (CID) at 20 keV. Using both protonated and sodiated ions, this technique enabled us to propose general rules to discriminate between isomeric and isobaric ceramide species, unrelated to the presence or absence of sugar chains. In addition, this high-energy CID generated ^3,5A ions, indicating Hex1-4Hex linkage in the sugar chains. Using this method, we demonstrated distinct differences among ceramide species, including free ceramides, sphingomyelins, and neutral GSLs of glucosylceramides, galactosylceramides, lactosylceramides, globotriaosylceramides and Forssman glycolipids in the equine kidneys.     

Geographical distributions of mitochondrial DNA lineages reflect ancient directions of river flow: a case study of the Japanese freshwater shrimp Neocaridina denticulata denticulata (Decapoda: Atyidae)

The mitochondrial DNA phylogeographical structure of the freshwater atyid shrimp Neocaridina denticulata denticulata was investigated near the Yura and Kako Rivers in western Japan. To assess the biological significance of drainage evolution, fragments of 390 base pairs (bp) extending from the NADH dehydrogenase subunit 2 gene to the tryptophan transfer RNA gene were sequenced for 246 specimens from twenty locations. The phylogenetic tree detected two distinct clades corresponding to the region along the Sea of Japan and Seto Inland Sea, respectively. Geographical mapping of the two clades well reflected the river capture, in which the upper reaches of the Yura River draining into the Sea of Japan had been captured from the Kako River into the Seto Inland Sea. The clear pattern of mitochondrial DNA distribution may be accounted for by the ecological characteristics of species that minimize passive downstream drift, local adaptation, and mountain topography.     

Blocking synaptic transmission with tetanus toxin light chain reveals modes of neurotransmission in the PDF-positive circadian clock neurons of Drosophila melanogaster

Circadian locomotor rhythms of Drosophila melanogaster are controlled by a neuronal circuit composed of approximately 150 clock neurons that are roughly classified into seven groups. In the circuit, a group of neurons expressing pigment-dispersing factor (PDF) play an important role in organizing the pacemaking system. Recent studies imply that unknown chemical neurotransmitter(s) (UNT) other than PDF is also expressed in the PDF-positive neurons. To explore its role in the circadian pacemaker, we examined the circadian locomotor rhythms of pdf-Gal4/UAS-TNT transgenic flies in which chemical synaptic transmission in PDF-positive neurons was blocked by expressed tetanus toxin light chain (TNT). In constant darkness (DD), the flies showed a free-running rhythm, which was similar to that of wild-type flies but significantly different from pdf null mutants. Under constant light conditions (LL), however, they often showed complex rhythms with a short period and a long period component. The UNT is thus likely involved in the synaptic transmission in the clock network and its release caused by LL leads to arrhythmicity. Immunocytochemistry revealed that LL induced phase separation in TIMELESS (TIM) cycling among some of the PDF-positive and PDF-negative clock neurons in the transgenic flies. These results suggest that both PDF and UNT play important roles in the Drosophila circadian clock, and activation of PDF pathway alone by LL leads to the complex locomotor rhythm through desynchronized oscillation among some of the clock neurons.     

Excitatory backward conditioning in an appetitive conditioned reinforcement preparation with rats

Four experiments were conducted to examine appetitive backward conditioning in a conditioned reinforcement preparation. In all experiments, off-line classical conditioning was conducted following lever-press training on two levers. Presentations of a sucrose solution by a liquid dipper served as an unconditioned stimulus (US) and two auditory stimuli served as conditioned stimuli (CSs); one was paired with the US in either a forward (Experiment 1a) or a backward (Experiments 1b, 2, and 3) relationship, and the other served as a control CS, which was not paired with the US. In testing, each lever-press response produced a presentation of one of the CSs instead of appetitive reinforcers. The response to a lever was facilitated, compared to the response to another lever, when the response produced the backward CS presentation as well as when it produced the forward CS presentation; that is, the backward CS served as an excitatory conditioned reinforcer.     

A simple stereoselective synthesis and biological evaluation of FR181157: orally active prostacyclin mimetic

Synthetic method of novel prostaglandin (PG) mimetic: FR181175 without PG skeleton are described. The key to success is creation of a chiral epoxide using Sharpless AD reaction with high ee yield. FR181157 shows high potency and agonist efficacy at the IP receptor and has good bioavailability.     

Efficient expansion of mouse primary tenocytes using a novel collagen gel culture method

Development of regenerative therapies for damaged tendons remains a great challenge, largely because of lack of information regarding the mechanisms responsible for differentiation of tenocytes. Mouse tenocytes have not been fully characterized owing to the absence of efficient and reproducible methods for their in vitro expansion without losing phenotypic features. The objective of the study was to establish an improved and reliable method for stable primary culture of mouse tenocytes by using collagen gel. Achilles and tail tendon tissues were harvested and embedded in collagen gel. After 10 days of continuous culture, the gel was digested and cells were passaged on tissue culture-treated plastic dishes. Mouse tenocytes cultured in collagen gel exhibited significantly shorter doubling time and higher numbers of proliferation when maintained on the plastic dishes compared with those cultured without using gel. Transmission electron microscopic analyses showed that cultured tenocytes retained some morphological features of tenocytes in tendon tissues, such as cell–cell junctional complex formation, well-developed rough endoplasmic reticulum, and mitochondria in their cytoplasm. mRNA expression of tenocyte markers (tenomodulin, type I collagen, periostin, and scleraxis) was higher in cells cultured in collagen gel than in those cultured in the absence of gel. Our results show that tenocytes cultured using the collagen gel method express typical lineage markers and exhibit improved growth characteristics, thus providing a stable platform for studying molecular mechanisms that control their differentiation.