Development of an oesophageal stimulation method to elicit swallowing reflex in humans.

Swallowing reflex is known to be evoked by gastroesophageal regurgitation or oesophageal stimulation in animal studies. However, details regarding the stimulating material, bolus size and stimulation area remain unclear for the stimulation-induced type of swallowing reflex in humans. Here, we evaluated the effects of different kinds of stimulation via water and air injection of the oesophagus on the initiation of the swallowing reflex. Nine healthy individuals participated in this study. A fibre-optic endoscope was passed transnasally, and a thin catheter for injection was passed through the other side. The tip of the catheter was placed at the upper, upper middle, lower middle or lower region of the oesophagus, and the rate of injection was controlled at 0.2 mL/s. Swallowing reflex latency was calculated as the time from injection via air or thin/thick fluid until the onset of white-out in endoscopic images. Reflex latency was significantly shorter when injection occurred at the upper region of the oesophagus than at the lower region, for both thin and thick fluids (P < .01). At the upper region of the oesophagus, the latency was significantly shorter after injection of thin fluid than with thick fluid (P < .05). Injection of air did not induce the swallowing reflex at all sites. These findings suggest that while the swallowing reflex is evoked by stimulation via fluid injection of the oesophagus in humans, sensitivity is greatest in the upper region of the oesophagus compared with the lower region and can vary depending on the injecting material.

The effect of bolus volume on laryngeal closure and UES opening in swallowing: Kinematic analysis using 320-row area detector CT study.

This study investigated the effects of three different volumes of honey-thick liquid on the temporal characteristics of swallowing. Twenty-six healthy subjects (15 males, 11 females) underwent 320-row area detector CT scan while swallowing 3, 10 and 20 mL of honey-thick liquid barium. Three-dimensional images were created at 10 images/s. Kinematic events involving six structures (velopharynx, hyoid bone, epiglottis, laryngeal vestibule (LV), true vocal cords (TVC), upper esophageal sphincter (UES)) and timing of bolus movement were timed using frame by frame analysis. The overall sequence of events did not differ across three volumes; however, increasing bolus volume significantly changed the onset and termination of events. The bolus head reached to pharynx and esophagus earlier and the duration of bolus passing through UES was significantly longer in 10 and 20 mL compared to 3 mL (P < .05). Consequently, the onset of UES opening was significantly earlier with increased volume (P < .05). LV and TVC closure occurred later in 20 mL compared to 3 mL (P < .05). These changes in motion of pharynx and larynx appeared to promote swallow safety by preventing aspiration, suggesting that anatomical structure movements adapt in response to bolus volume. Our findings also suggest that the pharyngeal swallow behaviours may be modified by afferents in the oral cavity. The three-dimensional visualization and quantitative measurements provided by 320-ADCT provide essential benchmarks for understanding swallowing, both normal and abnormal.

Enhanced miR-210 expression promotes the pathogenesis of endometriosis through activation of signal transducer and activator of transcription 3.

STUDY QUESTION: What are the roles of the microRNA miR-210-an miRNA that is up-regulated in endometriotic cyst stromal cells (ECSCs)-in the pathogenesis of endometriosis? SUMMARY ANSWER: Up-regulated miR-210 expression in ECSCs is involved in their proliferation, resistance to apoptosis and angiogenesis through signal transducer and activator of transcription (STAT) 3. WHAT IS KNOWN ALREADY: In the pathogenesis of endometriosis, a number of roles for microRNAs (miRNAs) are becoming apparent. STUDY DESIGN, SIZE, DURATION: ECSCs and normal endometrial stromal cells (NESCs) were isolated from ovarian endometriotic tissues (patients aged 24-40 years undergoing salpingo-oophorectomy or evisceration for the treatment of ovarian endometriotic cysts, n = 10) and the eutopic endometrial tissues without endometriosis (premenopausal patients aged 35-45 years undergoing hysterectomies for subserousal leiomyoma, n = 13), respectively. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used a global gene expression microarray technique to identify downstream targets of miR-210, and we assessed the functions of miR-210 in the pathogenesis of endometriosis by using the miR-210-transfected NESCs. MAIN RESULTS AND THE ROLE OF CHANCE: Gene expression microarray analysis revealed that one of the key target molecules of miR-210 is STAT3. In the NESCs, in comparison to the control, miR-210 transfection resulted in the induction of cell proliferation (P < 0.0005), the production of vascular endothelial cell growth factor (VEGF) (P < 0.0005) and the inhibition of apoptosis (P < 0.05) through STAT3 activation [increased levels of mRNA (P < 0.0005), and protein (P < 0.005)]. In the ECSCs, inhibitors of STAT3 inhibited the cell proliferation and VEGF production (P < 0.05), and induced the apoptosis of these cells (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: The roles of aberrant miR-210 expression were investigated only in the stromal component of ectopic and eutopic endometrium. Control endometrial tissues were obtained from premenopausal patients who had subserosal leiomyoma and NESC gene expression patterns may be altered in these women. Furthermore, the effects of STAT3 inhibitors were evaluated only in ECSCs and not in NESCs. WIDER IMPLICATIONS OF THE FINDINGS: The present findings indicate that miR-210 induces NESCs to differentiate into the endometriotic phenotype and we speculate that up-regulated miR-210 expression in ECSCs is involved in the creation of the endometriosis-specific cellular dysfunctions through epigenetic mechanisms. The data indicate that STAT3 inhibitors may be promising candidates for the treatment of endometriosis. STUDY FUNDING/COMPETING INTERESTS: This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 13237327 to K.N., no. 25861500 to Y.K. and no. 23592407 to H.N.). There are no conflicts of interest to declare.

Effects of fermented milk intake and physical activity on the suppression of age-related decline in physical fitness among the elderly.

Physical deterioration in the elderly can lead to disability and mortality. Although the intake of fermented milk has been recently attracting attention as a proposed measure to prevent physical weakness, studies and findings are limited. Here, we investigated the effect of intake of fermented milk products on suppression of age-related decline in physical fitness through a long-term epidemiological study of community-dwelling elderly people who are capable of independent living. A retrospective analysis was conducted on 581 elderly people aged 65-92 years from the Nakanojo Study, with the addition of a 5-year prospective analysis on 240 elderlies. Subjects were arbitrarily grouped on the basis of questionnaire estimates of fermented milk products intake (<3 or ≥3 days/week) and pedometer/accelerometer-determined patterns of physical activity (<7,000 or ≥7,000 steps/day). After adjustment for potential confounders, the retrospective study showed that the group consuming fermented milk products ≥3 days/week showed significantly faster walking speeds than the <3 days/week group. The group taking ≥7,000 steps/day had a significantly faster walking speed than the group taking <7,000 steps/day. Those who did both walked the fastest, indicating an additive effect. Adding protein or energy intake as a covariate to the potential confounders found a correlation between the intake of fermented milk products and walking speed, suggesting that the effect of fermented milk products consumption is independent of nutritional intake status, due to the beneficial properties of bacteria included in fermented milk. The 5-year prospective study confirmed a clear relationship between the frequency of consumption of fermented milk products and the suppression of preferred walking speed decline. Our findings suggest that habitual intake of fermented milk contributes to the suppression of walking speed decline in elderly people.

Novel electric power-driven hydrodynamic injection system for gene delivery: safety and efficacy of human factor IX delivery in rats.

The development of a safe and reproducible gene delivery system is an essential step toward the clinical application of the hydrodynamic gene delivery (HGD) method. For this purpose, we have developed a novel electric power-driven injection system called the HydroJector-EM, which can replicate various time-pressure curves preloaded into the computer program before injection. The assessment of the reproducibility and safety of gene delivery system in vitro and in vivo demonstrated the precise replication of intravascular time-pressure curves and the reproducibility of gene delivery efficiency. The highest level of luciferase expression (272 pg luciferase per mg of proteins) was achieved safely using the time-pressure curve, which reaches 30 mm Hg in 10 s among various curves tested. Using this curve, the sustained expression of a therapeutic level of human factor IX protein (>500 ng ml(-1)) was maintained for 2 months after the HGD of the pBS-HCRHP-FIXIA plasmid. Other than a transient increase in liver enzymes that recovered in a few days, no adverse events were seen in rats. These results confirm the effectiveness of the HydroJector-EM for reproducible gene delivery and demonstrate that long-term therapeutic gene expression can be achieved by automatic computer-controlled hydrodynamic injection that can be performed by anyone.

Expanded polyglutamine stretches interact with TAFII130, interfering with CREB-dependent transcription.

At least eight inherited neurodegenerative diseases are caused by expanded CAG repeats encoding polyglutamine (polyQ) stretches. Although cytotoxicities of expanded polyQ stretches are implicated, the molecular mechanisms of neurodegeneration remain unclear. We found that expanded polyQ stretches preferentially bind to TAFII130, a coactivator involved in cAMP-responsive element binding protein (CREB)-dependent transcriptional activation, and strongly suppress CREB-dependent transcriptional activation. The suppression of CREB-dependent transcription and the cell death induced by polyQ stretches were restored by the co-expression of TAFII130. Our results indicate that interference of transcription by the binding of TAFII130 with expanded polyQ stretches is involved in the pathogenetic mechanisms underlying neurodegeneration.

Relationships between dietary diversity and gut microbial diversity in the elderly.

Diet is considered as a major driver of gut microbiota composition. However, little is known about the relationship between overall dietary balance and gut microbiota, especially in the elderly. Here, using the Quantitative Index for Dietary Diversity (QUANTIDD), we analysed the relationships between dietary diversity and gut microbiota diversity in 445 Japanese subjects aged 65-90 years. We also examined the effect of age by comparing the young-old group aged 65 to 74 years (<75 years group; n=246) and the old-old group aged 75 years and older (≥75 years group; n=199). QUANTIDD showed significant positive relationships with Pielou's evenness and Shannon indices, two α-diversity indices related to the uniformity of species distribution. This suggests that a more diverse diet is associated with a more uniform abundance of various bacterial groups, rather than a greater variety of gut bacteria. QUANTIDD also showed significant positive associations with the abundance of Anaerostipes, Eubacterium eligens group, and Eubacterium ventriosum group, which produce short-chain fatty acids (SCFAs) and are beneficial to health. Negative association was found with the abundance of Ruminococcus gnavus group, which produces inflammatory polysaccharides. Positive associations between QUANTIDD and α-diversity indices or the abundance of specific bacterial groups were identified among all subjects and in the <75 years group, but not in the ≥75 years group. Our results suggest that dietary diversity contributes to the diversity of the gut microbiota and increases the abundance of SCFAs-producing bacteria, but only up to a certain age. These findings help to understand the complex relationship between diet and gut microbiota, and provide hints for specific dietary interventions to promote beneficial gut microbiota in the elderly.

Expression profiles of genes associated with viral entry in HCV-infected human liver.

Recent studies have demonstrated that several cellular factors are involved in entry of hepatitis C virus (HCV) into host cells. Detailed gene expression profiles of these factors in HCV-infected livers have not been reported for humans. Transcriptional levels of LDL receptor (LDLR), CD81, scavenger receptor class B type I (SR-BI), claudin-1, and occludin genes in liver samples from patients with chronic hepatitis C were investigated. Serum levels of LDL-cholesterol (LDL-C) and HCV core antigen were also evaluated, and expression of claudin-1 and occludin were immunohistochemically analyzed. Compared with normal liver, transcription of LDLR and claudin-1 genes was significantly suppressed (P < 0.0001) and occludin transcription was significantly up-regulated in HCV-infected livers (P < 0.0001). Significant positive correlations were found for LDLR versus occludin, LDLR versus claudin-1, occludin versus claudin-1, and CD81 versus SR-BI in HCV-infected (P = 0.0012, P < 0.0001, P = 0.0004, and P < 0.0001, respectively) and normal livers (P < 0.0001, P = 0.0051, P < 0.0001, and P < 0.0001, respectively). Positive correlation was observed between serum levels of HCV core antigen and LDL-C (P = 0.0147), with their levels negatively correlated to LDLR (P = 0.0270 and P = 0.0021, respectively). Immunohistochemically, hepatocellular expression of claudin-1 and occludin was increased in HCV-infected livers. Different levels of expression were demonstrated at the mRNA and protein levels for occludin and claudin-1 in HCV-infected and normal livers. Correlation of elements associated with viral entry was comparable in HCV-infected and normal livers.

Association of a polymorphism of BTN2A1 with type 2 diabetes mellitus in Japanese individuals.

AIMS: We previously showed that the C→T polymorphism (rs6929846) of BTN2A1 was significantly associated with myocardial infarction in Japanese individuals by a genome-wide association study. Given that diabetes mellitus is an important risk factor for myocardial infarction, the association of rs6929846 of BTN2A1 with myocardial infarction might be attributable, at least in part, to its effect on susceptibility to diabetes. The purpose of this study was to examine the relation of rs6929846 of BTN2A1 to Type 2 diabetes mellitus. METHODS: A total of 8650 Japanese individuals from two independent subject panels were examined: Panel A comprised 1141 individuals with Type 2 diabetes and 3161 control subjects and panel B comprised 1664 individuals with Type 2 diabetes and 2684 control subjects. RESULTS: The chi-square test revealed that rs6929846 of BTN2A1 was significantly related to the prevalence of Type 2 diabetes in subject panel A (P = 0.0002) and subject panel B (P=0.006). Multivariable logistic regression analysis with adjustment for age, sex, body mass index and smoking status revealed that rs6929846 was significantly associated with Type 2 diabetes (P = 0.0006; odds ratio 1.25) in all individuals, with the T allele representing a risk factor for this condition. Multiple regression analysis with adjustment for age, sex and body mass index revealed that rs6929846 was significantly (P=0.04) related to blood glycosylated haemoglobin content in control subjects. CONCLUSIONS: BTN2A1 may be a susceptibility gene for Type 2 diabetes in Japanese individuals.

Two cases of diverticulitis in patients with Williams syndrome.

Williams syndrome is rare and associated with physical anomalies and mental retardation. It is a disease resulting from a gene deletion of chromosome 7. The main concurrent medical conditions typically associated with Williams syndrome are heart defects such as supravalvular aortic stenosis, mental retardation, and unusual physical characteristics. It is also associated with colon diverticulosis and diverticulitis. In the present article, we report on 2 cases of diverticulitis in patients with Williams syndrome, in whom surgery was performed. In many cases of diverticulitis in patients with Williams syndrome, surgical treatment is indicated. It is important to take diverticulitis into consideration when examining a patient with Williams syndrome presenting with abdominal pain and consider surgical treatment if necessary.

A case of fulminant amebic colitis with multiple large intestinal perforations.

Amebic colitis normally causes mucous and bloody diarrhea stool as predominant symptoms, thus leading to a course of chronic colitis. However, though rare, there exists a fulminating type that causes intestinal perforations due to wide necrosis of the large intestine. We encountered a case of fulminant amebic colitis that lead to death due to multiple large intestinal perforations. The patient was a 72-year-old female. The patient was admitted to our hospital with symptoms of fever, abdominal pain, and diarrhea. She continued to have a fever of over 38 degrees C and increased left abdominal pain. An abdominal computed tomography scan revealed free gas on the abdominal side of the kidney. Therefore, gastrointestinal perforations were diagnosed and surgery was performed. In surgery, many perforated parts were observed from the appendix to the descending colon, and subtotal colectomy was performed. However, sepsis and disseminated intravascular coagulation occurred, and the patient died on the eighth postoperative day.

Genetic risk for metabolic syndrome: examination of candidate gene polymorphisms related to lipid metabolism in Japanese people.

BACKGROUND: The aetiology of metabolic syndrome is complex, being determined by the interplay of both genetic and environmental factors. The aim of this study was to identify genetic polymorphisms that confer susceptibility to metabolic syndrome, to allow prediction of genetic risk for this condition. METHODS: The study population comprised 2417 unrelated Japanese subjects (1522 with metabolic syndrome and 895 controls). The genotypes for 44 polymorphisms of 31 candidate genes related to lipid metabolism were determined using a combination of PCR and sequence-specific oligonucleotide probes with suspension array technology. RESULTS: The chi(2) test and subsequent multivariate logistic regression analysis with adjustment for age, sex and smoking status found that the-3A-->G and 553G-->T (Gly185Cys) polymorphisms of APOA5, the 2052T-->C (Val653Val) and 1866C-->T (Asn591Asn) polymorphisms of LDLR, the 13989A-->G (Ile118Val) polymorphism of CYP3A4 and the 1014T-->A polymorphism of C1QTNF5 were significantly (false discovery rate <0.05) associated with the prevalence of metabolic syndrome, with the variant alleles of APOA5 and C1QTNF5 representing risk factors for and those of LDLR and CYP3A4 being protective against this condition. Serum levels of triglycerides and high-density lipoprotein (HDL) cholesterol differed significantly (p<0.05) among APOA5 genotypes; the serum level of HDL cholesterol differed among LDLR genotypes; and the fasting plasma glucose level and body mass index differed between CYP3A4 and C1QTNF5 genotypes, respectively. CONCLUSIONS: APOA5, LDLR, CYP3A4 and C1QTNF5 are susceptibility loci for metabolic syndrome in Japanese people. Genotypes for these polymorphisms may prove informative for prediction of genetic risk for metabolic syndrome.

Genetic risk for myocardial infarction determined by polymorphisms of candidate genes in a Japanese population.

BACKGROUND: Although several environmental factors influence the development of myocardial infarction (MI), genetic factors have been shown to contribute to individual susceptibility to this condition. OBJECTIVE: To identify gene polymorphisms that confer susceptibility to MI in order to allow assessment of genetic risk for this condition. METHODS: 3433 unrelated Japanese people (1931 men, 1502 women) were entered into the study. These comprised 1328 subjects with MI (1036 men, 292 women) and 2105 controls (895 men, 1210 women). The genotypes for 40 polymorphisms of 31 candidate genes were determined with a method that combines PCR and sequence-specific oligonucleotide probes with suspension array technology. RESULTS: The chi(2) test revealed that six polymorphisms were significantly (false discovery rate <0.05) related to the prevalence of MI. Further examination by multivariable logistic regression analysis with adjustment for age, sex, body mass index and the prevalence of hypertension, diabetes mellitus and hypercholesterolaemia, in addition to a stepwise forward selection procedure found that the A-->C (Gln1334His) polymorphism (rs3742207) of the collagen type IV alpha-1 gene (COL4A1) and the A-->G polymorphism (rs4804611) of the zinc finger protein 627 gene (ZNF627) were significantly (p<0.05) associated with the prevalence of MI. The variant C allele of COL4A1 was protective against MI, whereas the variant G allele of ZNF627 represented a risk factor for this condition. CONCLUSIONS: Determination of genotypes for COL4A1 and ZNF627 may prove informative for assessment of the genetic risk for MI.

Superovulation and embryo transfer in Holstein cattle using sexed sperm.

The use of sexed bull sperm in multiple ovulation and embryo transfer (MOET) programs for Holsteins was evaluated for (1) heifers housed at a commercial embryo transfer (ET) facility (Experiments 1 and 2), and (2) heifers and cows on dairy farms (Experiment 3). In Experiment 1, superstimulated heifers were inseminated with 5 x10(6) sexed (X-sorted; n=5) or unsexed (n=5) frozen-thawed sperm from one bull at 12 and 24h after estrus detection. No difference was observed in the rates of transferable embryos (53.4% vs 68.1%), degenerate embryos (24.8% vs 26.6%) and unfertilized ova (21.8% vs 5.3%) between sexed and unsexed sperm, respectively, except for the percent of female transferable embryos diagnosed by embryo sexing (100% vs 49.3%, P<0.0001). In Experiment 2, donors were inseminated twice with 5 x10(6) sexed unfrozen sperm (n=10) or sexed frozen-thawed sperm (n=9). Embryo production rates for both treatments were similar to that observed on a commercial ET facility using unsexed sperm. Pregnancy rates for frozen-thawed embryos were similar for sexed and unsexed sperm (70.4% vs 72.4%, respectively). In Experiment 3, 99 flushes were conducted using sexed frozen-thawed sperm from nine bulls but an overall statistical analysis was not completed because the use of bulls was not balanced. However, for one bull with balanced usage, the rate of transferable embryos was higher in heifers than in cows (P<0.05) inseminated twice with 5 x10(6) sperm/dose (10 x10(6) total). We concluded that the use of sexed frozen-thawed sperm (> or =90% X-sperm biased and 10 x10(6) total sperm) may be economically viable for commercial MOET programs in Holstein heifers.

Lack of Bcl11b tumor suppressor results in vulnerability to DNA replication stress and damages.

Bcl11b/Rit1 is involved in T-cell development and undergoes chromosomal rearrangements in human T-cell leukemias. Thymocytes of Bcl11b(-/-) newborn mice exhibit apoptosis at a certain developmental stage when thymocytes re-enter into the cell-cycle. Here, we show that Bcl11b-knockdown T-cell lines, when exposed to growth stimuli, exhibited apoptosis at the S phase with concomitant decreases in a cell-cycle inhibitor, p27 and an antiapoptotic protein, Bcl-xL, owing to transcriptional repression. This repression was a likely consequence of the impairment of Sirt1, a nicotinamide adenine dinucleotide-dependent deacetylase associating with Bcl11b. Activation of the apoptotic process cleaved the mediator protein, Claspin, and inhibited phosphorylation of cell-cycle checkpoint kinase 1 (Chk1) that plays a central role in sensing and responding to incomplete replication. Bcl11b(-/-) thymocytes also failed to phosphorylate Chk1 when UV irradiated. These results implicate Bcl11b in the remedy for DNA replication stress and maintenance of genomic integrity.

Economic impact of extended treatment with peginterferon alpha-2a and ribavirin for slow hepatitis C virologic responders.

It is difficult to achieve a sustained virologic response from antiviral therapy for genotype 1 hepatitis C virus-infected patients without a sufficient virologic response in the early weeks after treatment. However, a recent study has reported on the effectiveness of an extended course of treatment with peginterferon alpha-2a plus ribavirin for slow virologic responders. The aim of this study was to evaluate the economic impact of an extended course of treatment. A Markov cohort model of hepatitis C was designed in order to demonstrate the clinical states, based on the assigned transition probabilities over 30 years. The slow virologic responders treated with an extended 72-week course of therapy could increase by 0.55 the quality-adjusted life years (=15.35-14.80) and reduce the lifetime cost by $2762 (=71 559-69 438) in comparison with those treated by the standard 48-week course. One-way sensitivity analyses did not change the cost-effectiveness. Therefore, the extended 72 weeks of treatment with peginterferon alpha-2a plus ribavirin for slow virologic responders could be cost-effective in comparison with the standard 48 weeks of treatment.

Habitual intake of fermented milk products containing Lactobacillus casei strain Shirota and a reduced risk of hypertension in older people.

This study investigated relationships between the frequent intake of fermented milk products containing Lactobacillus casei strain Shirota (LcS) and the onset of hypertension (resting systemic pressure ≥140 mmHg [systolic]/≥90 mmHg [diastolic], a doctor's diagnosis and/or antihypertensive medicine use) during a 5-year period in 352 communityliving Japanese aged 65 to 93 years (125 men and 227 women). Initially normotensive subjects were divided into two groups (n=254 and n=98) on the basis of their intake of fermented milk products (<3 or ≥3 times/week, respectively), as estimated during an interview by a certified nutritionist. The incidence of hypertension over the 5-year interval was significantly lower in those who took fermented milk products ≥3 rather than <3 times/week (6.1 vs 14.2%, P=0.037). A multivariate-adjusted proportional hazards model predicted that blood pressures were significantly more likely to remain normal over 5 years in subjects who took ≥3 fermented milk products rather than <3 times/ week (relative risk 0.398 [95% confidence interval 0.167-0.948], P=0.037). These results suggest that after adjustment for potential confounders, the risk of developing hypertension is substantially lower in elderly people who take fermented milk products containing LcS at least 3 times a week.

Comparison between human-telomerase reverse transcriptase mRNA and alpha-fetoprotein mRNA as a predictive value for recurrence of hepatocellular carcinoma in living donor liver transplantation.

OBJECTIVE: In this study we compared the potential roles of preoperative human-telomerase reverse transcriptase (h-TERT) mRNA versus alpha-fetoprotein (AFP) mRNA expression in the peripheral blood as a tool to predict prognosis and tumor recurrence after living donor liver transplantation (LDLT) in hepatocellular carcinoma (HCC) patients. PATIENTS AND METHODS: We examined 14 patients with unresectable HCC who underwent LDLT. Six patients displayed stage IVA HCC that deviated from the Milan criteria, while the rest of the patients fell within the limitations of the Milan criteria. We analyzed the relationship between preoperative h-TERT mRNA or AFP mRNA expression in the peripheral blood and survival without recurrence. RESULTS: There was no significant difference between the survival curves without recurrence of those patients who did versus did not meet the Milan criteria. There was also no significant difference between the survival curves without recurrence among patients with positive versus negative AFP mRNA expression. However, there was a significant difference (P = .005) between the survival curves without recurrence of those patients with positive preoperative h-TERT mRNA expression versus those who either had an initially negative preoperative h-TERT mRNA or who converted from positive to negative after neoadjuvant immunochemotherapy. CONCLUSIONS: h-TERT mRNA seemed to prove more valuable than AFP mRNA not only to assess preoperative treatment modalities and postoperative patient surveillance, but also to evaluate prospective LDLT patients with HCC. Moreover, use of h-TERT mRNA could potentially expand the indications for transplantation to patients outside the Milan criteria.

alpha-Fetoprotein as a carrier protein in plasma and its bilirubin-binding ability.

The bilirubin-binding ability of human alpha-fetoproteins, which were purified from fetal cord serum and from ascites fluid of a hepatoma-bearing patient, was examined by the difference spectrum and the Jacobsen peroxidase methods. The difference spectrum observed as a result of the specific binding of bilirubin to alpha-fetoprotein had a maximum at 482 nm, and this pattern was quite similar to that observed for serum albumin. The result obtained by the difference spectrum method showed that 1 mol of each alpha-fetoprotein bound 1 mol of bilirubin at pH 8.3 and that the dissociation constants of the complexes of bilirubin with fetal alpha-fetoprotein and hepatoma-derived alpha-fetoprotein were 2.6 x 10(-7) and 5.0 x 10(-7) M, respectively. The Jacobsen enzymatic method using horseradish peroxidase gave the same values for molar binding ratios and similar dissociation constants, 7.1 x 10(-7) M for fetal alpha-fetoprotein and 7.4 x 10(-7) M for hepatoma-derived alpha-fetoprotein. These results indicate that alpha-fetoprotein may function as a carrier protein for bilirubin as has been shown for serum albumin.

Comparative chemical structures of human alpha-fetoproteins from fetal serum and from ascites fluid of a patient with hepatoma.

Human alpha-fetoproteins were purified from umbilical cord serum and from ascites fluid of a patient with hepatoma by affinity chromatography, and their chemical compositions and terminal sequences were compared. The amino acid compositions of these alpha-fetoproteins were similar and in good agreement with the values reported by other investigators. The COOH-terminal 5-amino acid sequence determined by carboxypeptidase digestion and the NH2-terminal 20-amino acid sequence determined by an automated sequence analyzer revealed that both alpha-fetoproteins had the same terminal sequences of amino acids. The sequence analysis showed that a part of each of the proteins lacked its NH2-terminal residues for one or three amino acids. A small difference in the carbohydrate composition of each alpha-fetoprotein was observed. It was concluded that alpha-fetoproteins from fetal serum and from ascites fluid of a patient with hepatoma had very similar structures.