Preoperative intra-arterial chemotherapy with docetaxel, cisplatin, and peplomycin combined with intravenous chemotherapy using 5-fluorouracil for oral squamous cell carcinoma.

The objectives of this study were to evaluate survival in 141 patients with stage II-IV oral squamous cell carcinoma (OSCC) treated with preoperative intra-arterial chemotherapy with docetaxel, cisplatin, and peplomycin combined with intravenous chemotherapy using 5-fluorouracil (IADCPIVF) via the superficial temporal artery, and to clarify the prognostic factors. The study population included 59 patients with stage II OSCC, 34 with stage III, and 48 with stage IV. After IADCPIVF, 139 patients underwent surgery; minimally invasive surgeries (MIS) including excisional biopsy were performed on 96 patients with a remarkably good response to IADCPIVF. The primary tumour response rate was 99.3% (complete response rate 56.7%, good partial response rate 17.0%, fair partial response rate 25.5%). Additionally, there were no serious adverse events associated with IADCPIVF. The 5-year overall survival rate was 74.6% (stage II 83.6%, stage III 72.7%, stage IV 64.8%). In the multivariate analysis of survival, T classification and clinical tumour response were significant prognostic factors. Eight (8.3%) of the patients who received MIS had primary recurrence and six were salvaged. In conclusion, IADCPIVF is safe and efficacious for treating OSCC, and MIS could reduce the extent of primary tumour resection in the case of a remarkably good response.

Astaxanthin improves the developmental competence of in vitro-grown oocytes and modifies the steroidogenesis of granulosa cells derived from bovine early antral follicles.

In this study we investigated the effect of astaxanthin (Ax), which exhibits strong antioxidant activity, during invitro growth (IVG) on the developmental competence of oocytes and steroidogenesis of granulosa cells derived from early antral follicles. Bovine oocyte-cumulus-granulosa complexes collected from early antral follicles were cultured for 12 days in the presence or absence (control) of 500µM Ax. The viability of oocytes and antrum formation in the granulosa cell layer during IVG culture were greater in the presence than absence of Ax (P<0.05). Regardless of Ax treatment, 17ß-oestradiol production increased during IVG culture; however, progesterone production was significantly lower in the presence than absence of Ax (P<0.05). Reactive oxygen species levels were lower in Ax-treated oocytes than in controls after IVG (P<0.05). Although nuclear maturation and cleavage rates did not differ between the Ax-treated and control groups, Ax treatment led to weaker cathepsin B activity in oocytes and better blastocyst rates than in controls (P<0.05). Accordingly, Ax treatment during IVG increased the total number of cells in blastocysts (P<0.05). These results indicate that Ax supplementation of IVG medium improves the quality of bovine oocytes due to its antioxidative effects on growing oocytes and its suppression of the luteinisation of granulosa cells.

Isotype-specific selection of high affinity memory B cells in nasal-associated lymphoid tissue.

Mucosal immunoglobulin (Ig)A dominance has been proposed to be associated with preferential class switch recombination (CSR) to the IgA heavy chain constant region, Calpha. Here, we report that B cell activation in nasal-associated lymphoid tissue (NALT) upon stimulation with the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to chicken gamma globulin caused an anti-NP memory response dominated by high affinity IgA antibodies. In the response, however, NP-specific IgG(+) B cells expanded and sustained their number as a major population in germinal centers (GCs), supporting the view that CSR to IgG heavy chain constant region, Cgamma, operated efficiently in NALT. Both IgG(+) and IgA(+) GC B cells accumulated somatic mutations, indicative of affinity maturation to a similar extent, suggesting that both types of cell were equally selected by antigen. Despite the selection in GCs, high affinity NP-specific B cells were barely detected in the IgG memory compartment, whereas such cells dominated the IgA memory compartment. Taken together with the analysis of the V(H) gene clonotype in GC and memory B cells, we propose that NALT is equipped with a unique machinery providing IgA-specific enrichment of high affinity cells into the memory compartment, facilitating immunity with high affinity and noninflammatory secretory antibodies.

Structures of the N-linked sugar chains in PAS-7 glycoprotein sharing the same protein core with PAS-6 glycoprotein from the bovine milk fat globule membrane.

Glycoproteins PAS-6 (50 kDa) and -7 (47 kDa) from the bovine milk fat globule membrane share a common protein core but differ in their carbohydrate moiety. We here analyzed and proposed the structures of the N-linked sugar chains of PAS-7. The N-linked sugar chains were liberated from PAS-7 by hydrazinolysis and, after modifying the reducing ends with 2-aminopyridine (PA), were separated into one neutral (7N, 55%) and two acidic (7M, mono-, 43%; 7D, di-, 2%) sugar chain groups. The latter were converted into neutral groups (7MN and 7DN) by sialidase digestion. 7N was finally separated into 5 chains (7N1A, 7N1B-1, 7N1B-2, 7N2A, and 7N2B), and 7MN and 7DN were separated into 3 (7MN1, 7MN2, and 7MN3) and 2 (7DN1 and 7DN2) chains, respectively. The structure of each of these PA-neutral sugar chains was determined by sugar analysis, sequential exoglycosidase digestion, partial acetolysis, and 1H-NMR spectroscopy. The results show that the 10 sugar chains were of the biantennary complex type with and without fucose. The structure of 7N2A, one of the major sugar chains, was proposed as; [structure: see text] A structural comparison between PAS-6 and -7 indicated that, although they shared the same protein core, their sugar moiety was markedly different, involving the existence of a different pathway during the post-transcriptional modification.

Purification of membrane-bound lactoferrin from the human milk fat globule membrane.

Although lactoferrin is known as a basic soluble glycoprotein, the presence of the membrane-bound form of this protein has also been demonstrated in human milk. Membrane-bound lactoferrin was extracted from the human milk fat globule membrane with a detergent mixture of 1% Tween-20, 0.5% C12E8, and 0.5 M KCl in 20 mM Tris-HCl (pH 7.4). Lactoferrin in the detergent-soluble fraction was purified by affinity chromatography with Concanavalin A and by hydrophobic chromatography with phenyl-Superose. The purified protein gave a single band of 80 kDa by SDS-PAGE. Its N-terminal amino acid sequence was consistent with that of human lactoferrin.

Natural polyreactive immunoglobulin A antibodies produced in mouse Peyer's patches.

To understand the biological function of natural immunoglobulin A (IgA) antibodies in Peyer's patches (PP), we generated IgA monoclonal antibody (mAb) clones from the PP of normal, unimmunized, specific pathogen-free BALB/c mice and examined their reactivities by enzyme-linked immunosorbent assay (ELISA). Many of these antibodies reacted with more than one antigen examined, suggesting that they were polyreactive Abs. Two mAbs agglutinated several different strains of commensal bacteria isolated from mice. To examine the genetic features of these polyreactive mAbs, the VH genes of seven different IgA mAbs were sequenced. The VH genes from the VGAM, J558 and 7183 families were compared with sequence from the mAbs with distinct VDJ rearrangements. One of the mAbs that agglutinated bacteria was encoded by a germline VH gene, but the VH region of the other polyreactive mAbs contained between seven and 11 mutated sites. No indication of antigenic selection was observed in the pattern of these mutated sites. Our results show that polyreactive IgA Abs are present in PP as a part of the normal B-cell repertoire. These polyreactive Abs may establish a natural immune homeostasis, and function as a polyreactive sensor to detect pathogenic invasion and to control immune response in the gut.

Local antibody response in Peyer's patches to the orally administered dietary protein antigen.

To understand local antibody production to dietary protein antigens in the gut, the reactivity of the monoclonal antibodies (mAbs) from Peyer's patches of BALB/c mice raised against orally administered hen egg lysozyme (HEL) was studied. These mAbs were of IgG1 (7 clones), IgA (5 clones) and IgM (13 clones) isotypes. Some of the HEL-binding mAbs preferentially reacted with reduced, carboxy-methylated HEL, rather than with native HEL. MAbs of the IgA and IgM isotypes had cross-reactivity with other unrelated environmental antigens such as E. coli, single-strand DNA, and soluble components of mouse food. In contrast, the IgG1 mAbs did not cross-react with these antigens. The average of the Kd values for HEL of these mAbs was in the order of 10(-6) M, which is moderately higher than those of mAbs from the preimmune repertoire. These results suggest that, under normal physiological conditions, orally administered dietary proteins predominantly induce the local production of polyreactive IgA/IgM antibodies cross-reacting with environmental luminal antigens.

Anti-DNA IgA autoantibodies are spontaneously generated in mouse Peyer's patches.

IgA antibodies in the mucosal immune system are produced specifically to environmental antigens such as virus and bacteria, and possibly to some food components, which will provide a potential luminal antigen, DNA. To study the immune response to DNA in the gut, we established B-cell hybridomas producing IgA monoclonal antibodies (mAb) from Peyer's patches (PP) of non-immunized, non-autoimmune, specific pathogen-free BALB/c mice, and examined their specificity by enzyme-linked immunosorbent assay (ELISA). Three mAb out of 18 bound strongly to self, bacterial and synthetic DNA, with Kd of about 10-7 m. One of the three mAb also reacted with the histone component and another reacted with some mouse food component. The VH genes of these three mAb have not previously been reported to have anti-DNA specificity, and carry putative somatically mutated sites favouring DNA binding in CDR. The features resemble those of anti-DNA antibodies found in human and murine models of systemic lupus erythmatosus (SLE), and are indicative of an antigen-driven selection process. Our findings suggest that even in normal healthy animals, anti-DNA antibodies of IgA isotype can be produced in certain peripheral environments such as in PP by spontaneous antigenic stimulation.

Structures of the N-linked sugar chains in the PAS-6 glycoprotein from the bovine milk fat globule membrane.

The structures of the N-linked sugar chains in the PAS-6 glycoprotein (PAS-6) from the bovine milk fat globule membrane were determined. The sugar chains were liberated from PAS-6 by hydrazinolysis, and the pyridylaminated sugar chains were separated into a neutral (6N) and two acidic chains (6M and 6D), the acidic sugar chains then being converted to neutral sugar chains (6MN and 6DN). 6N was separated into two neutral fractions (6N13 and 6N5.5), while 6MN and 6DN each gave a single fraction (6MN13 and 6DN13). The structure of 6N5.5, which was the major sugar chain in PAS-6, is proposed to be Man alpha1 --> 6 (Man alpha1 --> 3) Man beta1 --> 4GlcNAc beta1 --> 4GlcNAc-PA; 6N13, 6MN13 and 6DN13 are proposed to be Gal beta1 --> 3Gal beta1 --> 4GlcNAc beta1 --> 2Man alpha1 --> 6 (Gal beta1 --> 3Gal beta1 --> 4GlcNAc beta1 --> 2Man alpha1 --> 3) Man beta1 --> 4GlcNAc beta1 --> 4 (Fuc alpha1 --> 6)GlcNAc-PA; 6M and 6D had 1 or 2 additional NeuAc residues at the non-reducing ends of 6MN13 and 6DN13, respectively.

Gel Formation from Industrial Milk Whey Proteins under Hydrostatic Pressure: Effect of Hydrostatic Pressure and Protein Concentration.

The effects of high hydrostatic pressure and protein concentration on the denaturation and gelation of whey protein were investigated. Industrial whey protein isolate (WPI) and whey protein concentrate (WPC) solutions (pH 6.8) at various concentrations were pressurized for 10 min at 30 degrees C under 200-1000 MPa. With the WPI solution, the concentration for affecting the turbidity was 1% and was 6% for the viscosity at 400 MPa, while for inducing gelation, it was 10% at 600 MPa. With the WPC solution, the viscosity changed at a concentration >12%, and gel formation began at >18% at 400 MPa. The hardness and breaking stress of pressure-induced WPI gels increased with increasing concentration of WPI (12-18%) and hydrostatic pressure, the ratings for the 20% WPC gels being one-third those of the 20% WPI gels. The solubility of proteins from the pressure-induced WPI gels decreased with increasing pressure, while that of WPC gel induced at >600 MPa remained constant at approximately 50%. The microstructure of the WPI gels had a porous network form, whereas the WPC gels were irregular particulates. beta-Lactoglobulin, alpha-lactalbumin, and serum albumin preferentially participated in pressure-induced aggregation and gelation through S-S bonding.

Purification and characterization of novel whey glycoprotein WGP-88 which binds to a monoclonal antibody to PAS-4 glycoprotein in the bovine milk fat globule membrane.

A monoclonal antibody to the PAS-4 glycoprotein (78 kDa) of the bovine milk fat globule membrane (MFGM) specifically recognized PAS-4, and was named KAS4. A component recognized by KAS4 was found in whey protein, this being a glycoprotein of 88 kDa by SDS-PAGE and named WGP-88. WGP-88 was purified and characterized in comparison with PAS-4. WGP-88 had apparent pI values of 6.45 and 6.39, while those of PAS-4 were 7.39 and 7.35. Neuraminidase digestion shifted the pI values of WGP-88 to 6.57 and of PAS-4 to 7.52. WGP-88 was rich in polar amino residues (44.9 mol%), while PAS-4 was abundant in nonpolar amino acid residues (48.7 mol%). WGP-88 contained 17.1% of carbohydrate and PAS-4 had 7.2%. The results of reductive hydrolysis, N-glycanase digestion, and a lectin blot analysis suggested that N- and O-linked sugar chains were contained in both glycoproteins. WGP-88 and PAS-4 had a different N-terminal amino acid sequence. WGP-88 and PAS-4 respectively inhibited competitively the binding of KAS4 to PAS-4 and WGP-88. Our studies revealed WGP-88 recognized by KAS4 mAb to be a novel whey protein and to have different biochemical properties from those of PAS-4.

Rapid and simple procedure for purifying PAS-4 glycoprotein from bovine milk fat globule membrane.

The isolation and partial characterization of PAS-4 glycoprotein (78 kDa) from bovine milk fat globule membrane (MFGM) is described. PAS-4 was selectively extracted with Triton X-114 nonionic detergent and then fractionated on DEAE-Sepharose at pH 7.5. The PAS-4 fraction that was not bound on DEAE-Sepharose gave a single band by SDS-PAGE. The recovery of PAS-4 was 57.4% from MFGM. An amino acid analysis found a high percentage of nonpolar residues. Approximately 7.2% of carbohydrate from PAS-4 was composed of mannose, galactose (Gal), N-acetylglucosamine, N-acetylgalactosamine (GalNAc), and sialic acid, most of the Gal and GalNAc in PAS-4 being released after mild alkaline hydrolysis. This indicated that PAS-4 contained both N- and O-linked sugar chains in concordance with the results of lectin affinity. PAS-4 had apparent isoelectric points of 7.45, 7.41, and 7.32, but these were shifted to pI 7.47 by a neuraminidase treatment. The apparent molecular weight of PAS-4 after deglycosylation with N-glycanase was approximately 57,000 by SDS-PAGE.

Flow cytometric analysis of p53 expression during the cell cycle.

Mutant p53 expressed in many types of carcinoma lacks an inhibitory function on cell growth, but its role has been unclear. We performed two-parameter flow cytometry (FCM) to elucidate the relationship between the expression of p53 and the cell cycle in A431 cells. Fluorescence in situ hybridization proved that an A431 cell had two p53 genes whereas chromosome 17 was tetraploid. FCM showed that A431 cells expressed constantly high levels of p53 during the cell cycle. Under conditions of both serum deprivation and presence of hydroxyurea, p53 expression was decreased throughout the cell cycle, and the bivariate DNA/p53 distribution pattern during the cell cycle did not change. The expression of p53 was reduced to 60% for the first 4 h after the addition of cycloheximide, and showed no significant changes at least for 20 h. Treatment with Triton X-100 increased p53 immunoreactivity throughout the cell cycle. These results indicate that mutant p53 differs from proliferative markers such as PCNA, Ki-67 and DNA polymerase-alpha, and that there are no links between the expression of p53 and the cell cycle in A431 cells.

Purification and characterization of major glycoproteins, PAS-6 and PAS-7, from bovine milk fat globule membrane.

Two major glycoproteins (PAS-6 and PAS-7) from bovine milk fat globule membrane were selectively extracted with urea and KCl, co-purified by repeated gel filtration on Sephacryl S-200 and then separated by affinity chromatography on concanavalin A-agarose column. The two purified glycoproteins showed a single band by SDS-PAGE, and their molecular masses were estimated to be 50 kDa for PAS-6 and 47 kDa for PAS-7. Both PAS-6 and PAS-7 were resolved several variants by analytical isoelectric focusing. These were shifted to a single band at pI 6.2 for PAS-6 and at pI 6.5 for PAS-7 by neuraminidase. PAS-6 contained 7.1% and PAS-7 5.5% of carbohydrate; the molar ratio of fucose:mannose:galactose:N-acetyl galactosamine:N-acetyl glucosamine:sialic acid was 1.0:3.0:2.0:6.1:5.0:1.3 for PAS-6 and 1.0:3.1:2.2:0:4.1:1.1 for PAS-7. Mild alkaline treatment and affinity to various lectins indicated that PAS-6 had O- and N-linked oligosaccharide chains, while PAS-7 had only the N-linked type. The major amino acid residues of PAS-6 were Glu, Ser and Gly, and those of PAS-7 were Asp, Glu, Gly and Leu. The N-terminal amino acids of both glycoproteins were blocked. PAS-6 and PAS-7 digested with trypsin had a different peptide map, two major peptides having the same retention time on HPLC and being common to PAS-6 and PAS-7 having the same amino acid sequences of H-Gln-Ser-Gly-Asn-Lys-Asn-Pro-Ser-Glu-Ile-Ser-OH and H-Ile-Phe-Pro-Gly-Asn-Met-Asp-Asn-Ser-His-Lys-OH.

Enzymatic Properties of Two Calcium-stimulated ATPases in the Neutral and Acidic pH Regions Found in the Membrane Fraction of Human Milk.

Two calcium-stimulated ATPases at optimal pH values of 5.0 and 7.0, which are designated as acid and neutral Ca(2+) -ATPase, respectively, were found in the membrane fraction of human milk, and their enzymatic properties were studied. For maximal activity, neutral Ca(2+)-ATPase required 0.45 mM Ca ion, while acid Ca(2+) -ATPase required 207 mM Ca ion. Neutral Ca(2+) -ATPase activity was not enhanced by adding the Mg ion at more than 0.1 mM. Among the nucleotides, neutral Ca(2+) -ATPase showed a higher substrate specificity to GTP, CTP, ITP, and UTP than to ATP, while ATP was the best substrate for acid Ca(2+) -ATPase. Neutral and acid Ca(2+) -ATPases had apparent Km values of 0.361 and 0.192 mM, and Vmax of 186 and 178 µmol ATP hydrolyzed/mg of protein per min, respectively. Both Ca(2+) -ATPases were potently inhibited by fluoride, lanthanide, vanadate, and p-chloromercuribenzoate, and inactivated by EDTA, EGTA, and CDTA, but were unaffected by N-ethylmaleimide, NaN3, ouabain, oxidized glutathione, or oligomycin, and were inactivated by heating at 60°C for 10 min. These enzymes were concentrated in the membrane fraction of the cream and skim milk membrane.

Enzymic modification of alpha s1-casein with peptidylarginine deiminase: preparation of less acid-coagulable and less calcium-sensitive casein.

Enzymic modification with peptidylarginine deiminase (EC 3.5.3.15) enabled five out of six arginyl residues in alpha s1-casein to be converted to citrullyl residues, only the N-terminal arginyl residue remaining unaffected. An increase in the net negative charge was confirmed by PAGE. The isoelectric point was decreased from 4.46 for the intact alpha s1-casein to 4.30 for the deiminated type, while simultaneously lowering the acid-precipitation starting point from pH 5.17 to pH 4.62. The deiminated alpha s1-casein self-associated less in the absence of Ca and was less Ca-sensitive than the native type, although its Ca-binding ability was slightly enhanced. In the presence of 25 mM-CaCl2 and kappa-casein, Ca-induced precipitation of alpha s1-casein did not occur, the solution of the mixture remaining transparent. Deimination of alpha s1-casein resulted in altering its characteristics, possibly by interfering with interactions through hydrophobicity and/or hydrogen bonding. The positive charge of the arginyl residues might play an important role in casein micelle formation.

Decrease of opsin content in the developing rat photoreceptor cells by systemic administration of L-glutamate.

L-Glutamate, a putative photoreceptor cell neurotransmitter, causes thinning of the inner layers of the retina and has been used for preparing biologically fractionated photoreceptor cells. However, it is possible that absence of the inner retinal layers may affect the remaining retina, and/or glutamate may directly affect photoreceptor cells. We evaluated quantitatively the effects of L-glutamate on the developing photoreceptor cells by measuring the rod photoreceptor cell-specific protein, opsin. We purified rat rhodopsin and used it as the standard for measuring opsin content of rat retinas with competitive enzyme-linked immunosorbent assay. Various concentrations of glutamate were injected into 7-day-old rats, and the effects of the amino acid concentration on opsin expression were determined on postnatal day 14. Inner layers of the retina degenerated when 10 microliters or 15 microliters of 2.4 M glutamate/gram body weight was administered subcutaneously. Opsin content of these glutamate-treated retinas decreased significantly compared with control retinas. We administered glutamate to rats at various stages of development and determined the effects by light microscopy on postnatal day 14. The administration of glutamate resulted in no degeneration of the inner retina if injected on postnatal day 1 or 2, degeneration of the inner retina between day 3 to 7, and again, no degeneration after postnatal day 13. Opsin content decreased significantly when glutamate was administered between postnatal day 1 to 7, but not after day 13, the day the blood-retinal barrier seems to reach maturity. Our findings indicate that systemic administration of L-glutamate affects the expression of opsin in the developing rod photoreceptor cells.

Binding form of vitamin B2 in bovine milk: its concentration, distribution and binding linkage.

The contents of total, free, and bound vitamin B2 (B2) in bovine milk and their distribution in four separate milk fractions, including milk during the early lactation stage, were estimated. The total B2 content in whole mature milk was 179 +/- 25 micrograms/100 g (n = 16), and its distribution in the cream, whey, skim milk membrane, and casein fractions was 6, 67, 9, and 18%, respectively. The amount of flavins bound to protein in the total B2 was 13.6% in whole milk and rich in membrane fraction. The total B2 content (micrograms/100 g of milk) was higher in colostrum at 1-3 days (287 +/- 120) than in colostrum at 4-7 days (173 +/- 27), in transitional milk (182 +/- 33), and in mature milk (179 +/- 44). The bound flavin content decreased slightly as lactation progressed (20-30 micrograms/100 g), but the ratio of bound/total B2 did not vary (12-15%). Milk fat globule membrane (MFGM) contained 414 +/- 65 micrograms of B2/g of protein, most of it being bound to protein (92%). Market milks contained as much total B2 as raw whole milk, but the amount of bound form was only 2%. Guanidine HC1, urea, sodium dodecyl sulfate, pH at 3.0-3.5, delipidation, and boiling released most of the B2 bound to protein, suggesting that bound flavins bind to milk proteins by a hydrophobic linkage.

Transfer of orally administered alpha-tocopherol into human milk.

The transfer of orally administered alpha-tocopherol (1.1 g) into breast milk reached a maximum value of 414 micrograms/100 g milk, which was 6.6-fold the pre-supplemental level, after three days and declined to the base line level after five days. The amount of alpha-tocopherol recovered in the milk was 0.11%. The alpha-tocopherol equivalent/PUFA ratio (mg/g) was increased from 0.25 to values between 0.7 and 1.7 by the administration.