Relationship between general or specific immunoreactivity and prognosis in postoperative patients with hepatocellular carcinoma

Summary The levels of a variety of immunological parameters were examined in 203 preoperative patients with hepatocellular carcinoma (HCC) at various stages (I–IV). The changes in the peripheral blood lymphocyte (PBL) count, the serum level of immunosuppressive acidic protein and the degree of the skin reaction to purified protein derivative were associated significantly with the stage of HCC progression. However, the percentages of lymphocyte subsets, mitogenic responsiveness of PBL and serum immunoglobulin concentration remained at the levels of stage I. Further study demonstrated that in patients undergoing hepatic artery ligation, there were statistically significant correlations between the PBL count, immunosuppressive acidic protein concentration and intensity of the skin reaction to purified protein derivative, assayed 1 month after surgery, and the prognosis. HCC-specific immunity was examined in 34 patients treated by hepatic resection or hepatic artery ligation using in vitro responses of PBL to HCC extracts (ATS test). This test was performed using culture medium containing added arginine. None of the PBL from the patients showed a positive response to allogeneic HCC extracts, but the PBL from 12 patients (9 hepatic resections, 3 hepatic artery ligations) were stimulated significantly (SI 2.5) with autologous HCC extracts. In 7 of 9 hepatic resection patients who were positive in the ATS test, tumor recurrence was identified. Statistical analysis indicated that the ATS test result was significantly correlated with tumor recurrence in hepatic resection patients. Autologous-PBL-stimulating activities were isolated in a fraction at pH 8.3 and in fractions at pH 6.7–7.0 by chromatofocusing of the crude extract. Although identification of the HCC-specific antigen remains to be done, use of the above fractions may simplify the ATS test procedure and improve its sensitivity.     

Ascaris suum: suppression of reaginic and hemagglutinating antibody responses in the mouse by crude extract and maintenance fluid.

Intraperitoneal administration in mice of crude extract (CE) or maintenance fluid (MF) of Ascaris suum in Freund's incomplete adjuvant (FICA) in doses of 200 and 2 (CE) and 4 μg (MF) on Days ?4, 0, and +4 relative to the day of the immunization with 10 μg of hen egg white lysozyme (HL) resulted in the suppression of anti-HL reaginic antibody responses at varying degrees depending on the dose and their time of administration. Hemagglutinating antibody responses were also affected but in a different manner. Treatment with CE on Day ?4 resulted in complete suppression of reaginic antibody responses and some degree of suppression of hemagglutinating antibody responses depending on the size of the CE dose. In mice pretreated with MF, transient suppression was found only for reaginic antibody responses. In mice receiving the treatment of CE on Day 0, 200 μg of CE caused complete suppression of reaginic antibody responses, while 2 μg was less effective. Hemagglutinating antibody responses were also suppressed in proportion to the dose. Simultaneous treatment with MF did not cause any suppression of either reaginic or hemagglutinating antibody responses. In mice treated with CE on Day +4, reaginic antibody responses were not markedly suppressed and hemagglutinating antibody responses were also not altered. In contrast, treatment with MF on Day +4 resulted in suppression of reaginic antibody responses during the whole course of the primary response, but had no effect on hemagglutinating antibody responses. When MF was administered 7 days after the priming, no suppressive effect on the antibody responses was demonstrated. On the other hand, if a lower dose (1 μg of HL) was used for the priming, the effect of MF treatment with Day +4 was more pronounced in the primary reaginic antibody response and the secondary response was also affected. A comparable suppression of hemagglutinating antibody responses also was observed.     

Comparative studies on antibody formation against hen egg white lysozyme and its split fragment.

A peptide fragment Fr. 17 (Lys1-Cys-Asn27 Leu129-Cys-Ala122) of hen egg white lysozyme (HL) was previously found to retain at least one antigenic determinant region of the native protein. In this work a highly purified preparation of Fr. 17, contaminated with less than 0.01% HL and less than 1% of other fragments was found to be strongly immunogenic to rabbits. The kinetic patterns of antibody formation against Fr.17, assayed by passive hemagglutination (PHA), were quite different from those of antibody formation against HL. The specificity of the antibody elicited to Fr. 17 was mainly directed to the Fr. 9-10-a region (Ala11-Asn27) while that of the antibody elicited to the Fr. 17 region in native HL was directed to the Fr. 15-b region (Lys1-Cys-Ala10 Leu129-Cys-Trp123). It is concluded that in the process of antibody formation, the recognition of the Fr. 17 region in native HL is different from that of fragment Fr. 17.     

Dapagliflozin,a Sodium-Glucose Co-Transporter 2 Inhibitor,Acutely Reduces Energy Expenditure in BAT via Neural Signals in Mice

Selective sodium glucose cotransporter-2 inhibitor (SGLT2i) treatment promotes urinary glucose excretion, thereby reducing blood glucose as well as body weight. However, only limited body weight reductions are achieved with SGLT2i treatment. Hyperphagia is reportedly one of the causes of this limited weight loss. However, the effects of SGLT2i treatment on systemic energy expenditure have not been fully elucidated. Herein, we investigated the acute effects of dapagliflozin, a SGLT2i, on systemic energy expenditure in mice. Eighteen hours after dapagliflozin treatment oxygen consumption and brown adipose tissue (BAT) expression of ucp1, a thermogenesis-related gene, were significantly decreased as compared to those after vehicle treatment. In addition, dapagliflozin significantly suppressed norepinephrine (NE) turnover in BAT and c-fos expression in the rostral raphe pallidus nucleus (rRPa) which contains the sympathetic premotor neurons responsible for thermogenesis. These findings indicate that the dapagliflozin-mediated acute decrease in energy expenditure involves a reduction in BAT thermogenesis via decreased sympathetic nerve activity from the rRPa. Furthermore, common hepatic branch vagotomy abolished the reductions in ucp1 expression and NE contents in BAT and c-fos expression in the rRPa. In addition, alterations in hepatic carbohydrate metabolism, such as decreases in glycogen contents and upregulation of phosphoenolpyruvate carboxykinase, manifested prior to the suppression of BAT thermogenesis, e.g. 6 hours after dapagliflozin treatment. Collectively, these results suggest that SGLT2i treatment acutely suppresses energy expenditure in BAT via regulation of an inter-organ neural network consisting of the common hepatic vagal branch and sympathetic nerves.     

Novel (CA)n marker DXYS241 on the nonrecombinant part of the human Y chromosome

The origin of modern humans can be traced by comparing polymorphic sites in either mitochondria or genomic sequences between humans and other primates. The human Y chromosome has both a non-recombining region and X-Y homologous pseudo-autosomal regions. In the nonrecombining region events during evolution can be directly detected. At least a part of homology between Xq21 and Yp11 is a result of rather recent translocations from the X chromosome to the Y chromosome. DNA markers residing in the nonrecombining region of the human Y chromosome are potentially useful in tracing male-specific gene flow in human evolution. However, the number of available markers in the region is limited. Here, we report a novel X-Y homologous (CA)n repeat locus in the nonrecombining region of the Y chromosome. This marker, DXYS241, has several interesting features. Y- and X-chromosome alleles are distinguishable because the Y-chromosome alleles are shorter than the X-chromosome alleles most of the time. We developed 2 primer sets for specific examination of Y- and X-chromosome alleles. The marker should be useful in establishing relationships between populations based on patrilineal gene flow. Sequences homologous to DXYS241 are also found on the X chromosome of primates. Four events during primate evolution that led to the modern human Y chromosome were identified.     

Crystal structure of an ADP-dependent glucokinase from Pyrococcus furiosus: implications for a sugar-induced conformational change in ADP-dependent kinase

ADP-dependent kinases are used in the modified Embden-Meyerhoff pathway of certain archaea. Our previous study has revealed a mechanism for ADP-dependent phosphoryl transfer by Thermococcus litoralis glucokinase (tlGK), and its evolutionary relationship with ATP-dependent ribokinases and adenosine kinases (PFKB carbohydrate kinase family members). Here, we report the crystal structure of glucokinase from Pyrococcus furiosus (pfGK) in a closed conformation complexed with glucose and AMP at 1.9A resolution. In comparison with the tlGK structure, the pfGK structure shows significant conformational changes in the small domain and a region around the hinge, suggesting glucose-induced domain closing. A part of the large domain next to the hinge is also shifted accompanied with domain closing. In the pfGK structure, glucose binds in a groove between the large and small domains, and the electron density of O1 atoms for both the alpha and beta-anomer configurations was observed. The structural details of the sugar-binding site of ADP-dependent glucokinase were firstly clarified and then site-directed mutagenesis analysis clarified the catalytic residues for ADP-dependent kinase, such as Arg205 and Asp451 of tlGK. Homology search and multiple alignment of amino acid sequences using the information obtained from the structures reveals that eucaryotic hypothetical proteins homologous to ADP-dependent kinases retain the residues for the recognition of a glucose substrate.     

Characterization of the cupin-type phosphoglucose isomerase from the hyperthermophilic archaeon Thermococcus litoralis

The gene encoding phosphoglucose isomerase was cloned from Thermococcus litoralis, and functionally expressed in Escherichia coli. The purified enzyme, a homodimer of 21.5 kDa subunits, was biochemically characterized. The inhibition constants for four competitive inhibitors were determined. The enzyme contained 1.25 mol Fe and 0.24 mol Zn per dimer. The activity was enhanced by the addition of Fe(2+), but inhibited by Zn(2+) and EDTA. Enzymes with mutations in conserved histidine and glutamate residues in their cupin motifs contained no metals, and showed large decreases in k(cat). The circular dichroism spectra of the mutant enzymes and the wild type enzyme were essentially the same but with slight differences.