Purification, Rheological Characterization, and Visualization of Viscous, Neutral, Hetero-exopolysaccharide Produced by Lactic Acid Bacteria.

Viscous exopolysaccharide (EPS)-producing lactic acid bacteria (LAB) have received increasing interest in the dairy industry because of their capability to improve the texture and mouthfeel of fermented dairy products. To date, enormous efforts have been made to reveal the relationship between texture and EPS production in fermented milk products such as yogurt. However, the structure-rheology relationship of EPSs themselves is not yet well understood due to their low yields in general and their wide variety of chemical structures. In this chapter, we describe common techniques for the purification, visualization, and rheological analysis of viscous EPSs produced by LAB.

A naturally occurring α(s1)-casein-derived peptide in bovine milk inhibits apoptosis of granulosa cells induced by serum-free conditions.

Several naturally occurring peptides in bovine milk were characterized by tandem mass spectrometry and Edman degradation. Chromatograms of peptide fractions (passed through an ultra-filtration membrane, nominal molecular weight limit 3000) prepared from colostrum (collected immediately after parturition) and transitional milk (collected 5 days postpartum) showed that they were almost identical. In total, six peptides, α(s1)-CN (f16-23) (RPKHPIKH), α(s1)-CN (f16-24) (RPKHPIKHQ), α(s1)-CN (f17-25) (PKHPIKHQG), α(s1)-CN (f46-52) (VFGKEKV), α(s1)-CN (f94-105) (HIQKEDVPSER), and ß-CN (f121-128) (HKEMPFPK), were identified. One of the major peptides, the N-terminal fragment of αs1 -casein, varied structurally during early lactation: α(s1)-CN (f17-25) (PKHPIKHQG) and α(s1)-CN (f16-23) (RPKHPIKH)/α(s1)-CN (f16-24) (RPKHPIKHQ) were found in colostrum and transitional milk, respectively. A chemically synthesized peptide, α(s1)-CN (f16-23) (RPKHPIKH), inhibited apoptosis of bovine granulosa cells induced by serum-free conditions in a dose-dependent manner, in consequence of caspase-3 and caspase-9 suppressions. The physiological function of the peptide remains unclear, but it may have potential use as pharmaceutical agent and as an anti-apoptotic agent in cell culture medium.

Evolution of milk oligosaccharides and lactose: a hypothesis.

Mammalian milk or colostrum contains up to 10% of carbohydrate, of which free lactose usually constitutes more than 80%. Lactose is synthesized within lactating mammary glands from uridine diphosphate galactose (UDP-Gal) and glucose by a transgalactosylation catalysed by a complex of ß4-galactosyltransferase and α-lactalbumin (α-LA). α-LA is believed to have evolved from C-type lysozyme. Mammalian milk or colostrum usually contains a variety of oligosaccharides in addition to free lactose. Each oligosaccharide has a lactose unit at its reducing end; this unit acts as a precursor that is essential for its biosynthesis. It is generally believed that milk oligosaccharides act as prebiotics and also as receptor analogues that act as anti-infection factors. We propose the following hypothesis. The proto-lacteal secretions of the primitive mammary glands of the common ancestor of mammals contained fat and protein including lysozyme, but no lactose or oligosaccharides because of the absence of α-LA. When α-LA first appeared as a result of its evolution from lysozyme, its content within the lactating mammary glands was low and lactose was therefore synthesized at a slow rate. Because of the presence of glycosyltransferases, almost all of the nascent lactose was utilized for the biosynthesis of oligosaccharides. The predominant saccharides in the proto-lacteal secretions or primitive milk produced by this common ancestor were therefore oligosaccharides rather than free lactose. Subsequent to this initial period, the oligosaccharides began to serve as anti-infection factors. They were then recruited as a significant energy source for the neonate, which was achieved by an increase in the synthesis of α-LA. This produced a concomitant increase in the concentration of lactose in the milk, and lactose therefore became an important energy source for most eutherians, whereas oligosaccharides continued to serve mainly as anti-microbial agents. Lactose, in addition, began to act as an osmoregulatory molecule, controlling the milk volume. Studies on the chemical structures of the milk oligosaccharides of a variety of mammalian species suggest that human milk or colostrum is unique in that oligosaccharides containing lacto-N-biose I (LNB) (Gal(ß1 → 3)GlcNAc, type I) predominate over those containing N-acetyllactosamine (Gal(ß1 → 4)GlcNAc, type II), whereas in other species only type II oligosaccharides are found or else they predominate over type I oligosaccharides. It can be hypothesized that this feature may have a selective advantage in that it may promote the growth of beneficial colonic bacteria, Bifidobacteria, in the human infant colon.

Chemical characterization of the oligosaccharides in Bactrian camel (Camelus bactrianus) milk and colostrum.

Bactrian camel milk and colostrum are commonly used as foods in Mongolia, whose people believe that these products promote human health. It has been hypothesized that milk oligosaccharides are biologically significant components of human milk, acting as receptor analogs that inhibit the attachment of pathogenic microorganisms to the colonic mucosa, and as prebiotics, which stimulate the growth of bifidobacteria within the infant colon. To evaluate their biological significance, we studied the oligosaccharides present in samples of Bactrian camel milk and colostrum. Using (1)H-nuclear magnetic resonance spectroscopy, we identified and characterized the following oligosaccharides of camel colostrum: Gal(ß1-4)[Fuc(α1-3)]Glc (3-fucosyllactose), Gal(ß1-3)Gal(ß1-4)Glc (3'-galactosyllactose), Gal(ß1-6)Gal(ß1-4)Glc (6'-galactosyllactose), Neu5Ac(α2-3)Gal(ß1-4)Glc (3'-sialyllactose), Neu5Ac(α2-6)Gal(ß1-4)Glc (6'-sialyllactose), Neu5Ac(α2-3)Gal(ß1-3)Gal(ß1-4)Glc (sialyl-3'-galactosyllactose), Neu5Ac(α2-6)Gal(ß1-4)GlcNAc(ß1-3)Gal(ß1-4)Glc (sialyllacto-N-tetraose c), Neu5Ac(α2-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (sialyllacto-N-novopentaose a), Gal(ß1-3)[Neu5Ac(α2-6)Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (sialyllacto-N-novopentaose b); and Neu5Ac(α2-6)Gal(ß1-4)GlcNAc(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (monosialyllacto-N-neohexaose). The oligosaccharides in the mature camel milk were characterized as 3'-galactosyllactose, Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (lacto-N-novopentaose I), and 3'-sialyllactose.

Short communication: effect of grazing on the concentrations of total sialic acid and hexose in bovine milk.

Sialic acid, which is located at the terminal end of glycoconjugates, is believed to have important biological functions. Its concentration in bovine milk varies depending on lactation stage and season. However, it remains unclear whether dietary factors, especially fresh forage, affect the total sialic acid concentration in milk. The purpose of the present study was to investigate the effect of grazing on the concentrations of total sialic acid and hexose in bovine milk. Six healthy dairy cows were used in a crossover design (3 cows fed fresh forage and 3 cows fed grass silage) for 2 wk. Individual milk samples were collected at 2 consecutive milkings (morning and evening) at 0, 1, 3, 5, 8, 11, and 14 d of the experimental period, and 2 consecutive samples in each cow were combined on each sampling day in proportion of the morning and evening milk yields. No differences in body weight, milk yield, or milk composition were observed between the 2 groups during the experimental period. The hexose concentration in milk did not differ between these groups during the experimental period. Conversely, the total sialic acid concentration in the milk of each grazing cow significantly increased at 11 and 14 d of the experimental period compared with that at 0 d. In the grass silage group, the total sialic acid concentration at the end of the experimental period tended to be lower than that at 0 d, but the decrease was not significant. These results indicate that grazing management could have increased the concentration of sialoglycoconjugates in milk. This suggests that grazing may increase the biological function of milk because it is thought that sialic acid is significant in many ways.

Short communication: evidence for the presence of a putative odorant-binding protein in bovine colostrum.

Using a combination of PAGE and mass spectrometry for protein identification, we obtained evidence that a putative odorant-binding protein, designated hypothetical protein LOC517854, occurs in bovine colostrum. This protein, termed as a putative bovine colostral odorant-binding protein (bcOBP), consists of 172 AA residues, including a putative 16-AA signal peptide. The theoretical isoelectric point value and molecular mass of the full-length sequence of bcOBP were calculated to be 4.57 and 19604.18, respectively. The highest sequence similarity (83%) was observed with a potential pheromone transporter, Allergen Bos d 2. An odorant-binding protein derived from bovine nasal mucosa showed relatively low sequence similarity (52%) against bcOBP. Its biological function is unclear, but pheromone transport could be considered. This is the first report of a putative odorant-binding protein in bovine colostrum.

Structural characterization of oligosaccharides in the milk of an African elephant (Loxodonta africana africana).

The oligosaccharides present in the milk of an African elephant (Loxodonta africana africana), collected 4 days post partum, were separated by size exclusion-, anion exchange- and high-performance liquid chromatography (HPLC) before characterisation by (1)H NMR spectroscopy. Neutral and acidic oligosaccharides were identified. Neutral oligosaccharides characterised were isoglobotriose, Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc and a novel oligosaccharide that has not been reported in the milk or colostrum of any other mammal: Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc. Acidic oligosaccharides that are also found in the milk of Asian elephant were Neu5Ac(alpha2-3)Gal(beta1-4)Glc, Neu5Ac(alpha2-6)Gal(beta1-4)Glc, Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc, Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc, Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc and Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3){Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)}Gal(beta1-4)Glc, while Neu5Gc(alpha2-3)Gal(beta1-4)Glc, Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc, Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)[Gal(beta1-4)GlcNAc(beta1-6)]Gal(beta1-4)Glc and Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3){Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)}Gal(beta1-4)Glc have not been found in Asian elephant milk. The oligosaccharides characterised contained both alpha(2-3)- and alpha(2-6)-linked Neu5Ac residues. They also contain only the type II chain, as found in most non-human, eutherian mammals.

Variation of major neutral oligosaccharides levels in human colostrum.

OBJECTIVES: Human colostrum is known to be important for the protection of infants against infection by pathogenic microorganisms. This protection is thought to be due, partially, to various neutral and acidic oligosaccharides that are present in colostrum and milk. However, the concentrations of each of the oligosaccharide of human colostrum have not yet been determined. The aim of this present study was to determine the concentration of each of the major neutral oligosaccharide for three consecutive days from the start of lactation. METHOD: We analyzed the level of each neutral oligosaccharide in human colostrum, for three consecutive days from the start of lactation, obtained from 12 healthy Japanese women (ranging in age from 21 to 35 years; primipara 6 and multipara 6). The ABO blood groups of the donors were determined: A, three; B, three; O, five; AB, one. The determined human milk oligosaccharides were 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lactodifucotetraose (LDFT), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), three lacto-N-fucopentaose (LNFP I, II and III) and two lacto-N-difucohexaose (LNFDH I and II) using high-performance liquid chromatography (HPLC) with two derivatization techniques. RESULTS: The concentrations of 2'-FL and LDFT in colostrum on day 1 were significantly higher than those on days 2 and 3 (P<0.05). An increase in LNT was observed on day 3 compared with day 1 (P<0.05). CONCLUSION: These changes in concentrations of 2'-FL, LDFT and LNT may reflect the requirements for prebiotics and anti-infection agents by human infants during early lactation.

Selection of lactic yeast producing glucosylceramide from cheese whey.

From 2150 isolates from raw milk and milk products, yeast strains were surveyed to produce glucosylceramide from cheese whey. Most of the 54 strains that had accumulated a detectable amount of glucosylceramide were identified as Kluyveromyces lactis var. lactis. The cells of K. lactis var. lactis strain M-11 derived from domestic raw milk accumulated glucosylceramide 2.5-fold higher than K. lactis var. lactis NBRC 1267, the reference strain selected from the culture collections. Strain M-16 of K. lactis var. lactis derived from the same origin was found to synthesize a considerable amount of steryl glucoside in addition to glucosylceramide. Sequence analysis of ribosomal DNA intergenic spacer two regions revealed that strains M-11 and M-16 were diverged from a type strain of K. lactis var. lactis in the same species.

Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues.

MicroRNAs (miRNAs) are a non-coding family of genes involved in post-transcriptional gene regulation. These transcripts are associated with cell proliferation, cell differentiation, cell death and carcinogenesis. We analysed the miRNA expression profiles in 25 pairs of hepatocellular carcinoma (HCC) and adjacent non-tumorous tissue (NT) and nine additional chronic hepatitis (CH) specimens using a human miRNA microarray. Targets and references samples were co-hybridized to a microarray containing whole human mature and precursor miRNA sequences. Whereas three miRNAs exhibited higher expression in the HCC samples than that in the NT samples, five miRNAs demonstrated lower expression in the HCC samples than in the NT samples (P<0.0001). Classification of samples as HCC or NT by using support vector machine algorithms based on these data provided an overall prediction accuracy of 97.8% (45/46). In addition, the expression levels of four miRNAs were inversely correlated with the degree of HCC differentiation (P<0.01). A comparison of CH and liver cirrhosis samples revealed significantly different pattern of miRNA expression (P<0.01). There were no differences, however, between hepatitis B-positive and hepatitis C-positive samples. This information may help clarify the molecular mechanisms involved in the progression of liver disease, potentially serving as a diagnostic tool of HCC.

Concentrations of sialyloligosaccharides in bovine colostrum and milk during the prepartum and early lactation.

Sialyloligosaccharides and sialylglycoconjugates in colostrum and milk are regarded to be important biological components with respect to be source of brain gangliosides in infant and to be antiinfectional components for the attack by the pathogenic bacteria and virus. Several acidic oligosaccharides have been characterised in both bovine and human milk or colostrum. The sialyloligosaccharide content of human colostrum and milk has been extensively studied, whereas that of cows milk and colostrum has received less attention. In this study, the concentrations of three sialyloligosaccharides of bovine colostrum and milk were determined at various stages during the prepartum and the first 7 d postpartum. The concentration of 3'SL (Neu5Ac(alpha2-3)Gal(beta1-4)Glc) reached a maximum value of 0.85 mg/ml immediately following parturition while the concentrations of 6'SL (Neu5Ac(alpha2-6)Gal(beta1-4)Glc) and 6'SLN (Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc) of 0.14 and 0.12 mg/ml, respectively, were much lower at this initial stage, although these concentration were maximum immediately following parturition. Bovine colostrum, especially that collected immediately after parturition, may be suitable as a source of 3'SL and other sialyloligosaccharides for use as additives by the food or pharmaceutical industries.

Identification of human cancer-related genes by naturally occurring Hepatitis B Virus DNA tagging.

Proviral tagging has been used in animals as a powerful tool for cancer genetics. We show that a similar approach is possible in patients with hepatocellular carcinoma (HCC) infected by Hepatitis B Virus (HBV), a human pararetrovirus which may act by insertional mutagenesis. In this work, the HBV genome is used as a probe to identify cancer-related genes. By using HBV-Alu-PCR, we obtained 21 HBV/cellular DNA junctions from 18 different patients. In six of 21, we found the HBV DNA integrated into a cellular gene: (1) Sarco/Endoplasmic Reticulum Calcium ATPase1 Gene; (2) Thyroid Hormone Receptor Associated Protein 150 alpha Gene; (3) Human Telomerase Reverse Transcriptase Gene; (4) Minichromosome Maintenance Protein (MCM)-Related Gene; (5) FR7, a new gene expressed in human liver and cancer tissues; and (6) Nuclear Matrix Protein p84 Gene. Seven junctions contained unique cellular sequences. In the remaining eight, the HBV DNA was next to repetitive sequences, five of them of LINE1 type. The cellular genes targeted by HBV are key regulators of cell proliferation and viability. Our results show that studies on HBV-related HCCs allow to identify cellular genes involved in cancer. We therefore propose this approach as a valuable tool for functional cancer genomic studies in humans.

Effect of serum depletion on centrosome overduplication and death of human pancreatic cancer cells after exposure to radiation.

The tumor microenvironment is one of the key factors affecting the cellular response to radiation; however, the influence of serum concentration on tumor radiosensitivity remains poorly understood. We recently discovered that gamma-irradiation of tumor cells causes centrosome overduplication, which may lead to lethal nuclear fragmentation through the establishment of multipolar mitotic spindles. In the present study, we investigated the effect of serum depletion on radiation-induced cell death in relation to the centrosome dynamics in human pancreatic cancer cells. Exposure of Capan-1 cells to gamma-irradiation resulted in a time-dependent increase in cells containing multiple centrosomes in association with the appearance of mitotic cell death. Treatment of irradiated cells with serum depletion drastically accelerated centrosome overduplication and the formation of multipolar spindles, resulting in increased nuclear fragmentation and cell death. Cell cycle analysis of irradiated cultures revealed that the reduced serum level increased the population of cells arrested in the G2/M phase, which might be responsible for the abnormal centrosome accumulation. These findings suggest that serum concentration can influence radiation-induced cell killing through modulating cell cycle progression and possibly centrosome overduplication.

P21WAF1/CIP1 messenger RNA expression in hepatitis B, C virus-infected human hepatocellular carcinoma tissues.

BACKGROUND: The primary objective of this study was to clarify the significance of p21WAF1/CIP1(p21) gene expression in the tumorgenicity of hepatitis B virus (HBV) and hepatitis C virus (HCV) infected human hepatocelluar carcinoma (HCC). METHODS: The authors performed Northern blot hybridization to compare the p21 messenger (m) RNA expression levels among 16 HCC cases. They detected tissue HBVx mRNA (Northern blot) and plus- and minus-strand HCV RNA (reverse transcription-polymerase chain reaction) in liver tissues. They also measured alanine transaminase (ALT) levels and indocyanine green retention rate at 15 minutes (ICG-R15). RESULTS: The p21 transcripts of tumor (T) tissues could be identified with lower intensity than nontumor (N) tissues in all 4 HBVx mRNA(+) cases, 8 of 10 HCV RNA(+) cases, and 1 of 3 B(-), C(-) cases (1 case was positive for both viruses). p21 mRNA expression levels of N tissues were significantly higher in HCV RNA(+) cases than in HBVx mRNA(+) cases. p21 mRNA expression levels of N tissues were significantly correlated with serum ALT levels. CONCLUSIONS: In HCV hepatitis, p21 mRNA expression is up-regulated to control cell cycle under regeneration stress. Once the liver develops HCC, the p21 mRNA expression decreases to prominently low levels. The up-regulated p21 expression may play a role as a guard to prevent hepatocytes from tumorgenicity in HCV hepatitis.

Occurrence of an unusual phosphorylated N-acetyllactosamine in horse colostrum.

The colostrum of horses (thoroughbreds) was extracted and fractionated to yield Gal(beta1-4)GlcNAcalpha1-phosphate, which has not previously been detected in any mammalian milk or colostrum, as well as Neu5Ac(alpha2-3)Gal(beta1-4)Glc. The structures of these saccharides were established by NMR spectroscopy and MALDI-TOF mass spectrometry.

Possible role of telomerase activation in the multistep tumor progression of periampullary lesions in patients with familial adenomatous polyposis.

OBJECTIVES: The role of telomerase in periampullary tumor progression in patients with familial adenomatous polyposis (FAP) was investigated. METHODS: Relative telomerase activity was measured using a telomerease amplification protocol in periampullary biopsy specimens of normal mucosa and adenoma obtained from patients with FAP, and was compared with that of periampullary normal mucosa and cancer specimens from patients without FAP. RESULTS: None of normal mucosa from the non-FAP patients showed a telomerase ladder. Telomerase was positively detected in three of seven normal mucosa (42.9%) and in five of seven adenoma from FAP patients (62.5%). In papillary cancer from the non-FAP patients, seven of nine tissue specimens (77.8%) showed positive activity. When semiquantitatively analyzed, the relative telomerase activity increased in accordance with the progression of the diseases. CONCLUSIONS: Telomerase is activated even in normal mucosa of FAP patients, and the intensities of telomerase may reflect the malignant potential of periampullary neoplasms.

Chemical characterisation of the oligosaccharides in hooded seal (Cystophora cristata) and Australian fur seal (Arctocephalus pusillus doriferus) milk.

Carbohydrates were extracted from hooded seal milk, Crystophora cristata (family Phocidae). Free oligosaccharides were separated by gel filtration and then purified by ion exchange chromatography, gel filtration and preparative thin layer or paper chromatography and their structures determined by 1H-NMR. The hooded seal milk was found to contain inositol and at least nine oligosaccharides, most of which had lacto-N-neotetraose or lacto-N-neohexaose as core units, similar to those in milk of other species of Carnivora such as bears (Ursidae). Their structures were as follows: Gal(beta1-4)Glc (lactose); Fuc(alpha1-2)Gal(beta1-4)Glc (2'-fucosyllactose); Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (lacto-N-neotetraose); Fuc(alpha1-2)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (lacto-N-fucopentaose IV); Gal(beta1-4)GlcNAc(beta1-3)[Gal(beta1-4)GlcNAc(beta1-6)]Gal(1-4)Glc (lacto-N-neohexaose); Fuc(alpha1-2)Gal(beta1-4)GlcNAc(beta1-3)[Gal(beta1-4)GlcNAc(beta1-6)]Gal(beta1-4)Glc (monofucosyl lacto-N-neohexaose a); Gal(beta1-4)GlcNAc(beta1-3)[Fuc(alpha1-2)Gal(beta1-4)GlcNAc(beta1-6)]Gal(beta1-4)Glc (monofucosyl lacto-N-neohexaose b); Fuc(alpha1-2)Gal(beta1-4)GlcNAc(beta1-3)[Fuc(alpha1-2)Gal(beta1-4)GlcNAc(beta1-6)]Gal(beta1-4)Glc (difucosyl lacto-N-neohexaose); Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (para lacto-N-neohexaose); Fuc(alpha1-2)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (monofucosyl para lacto-N-neohexaose). Milk of the Australian fur seal, Arctophalus pusillus doriferus (family Otariidae) contained inositol but no lactose or free oligosaccharides. These results, therefore, support the hypothesis that the milk of otariids, unlike that of phocids, contains no free reducing saccharides.

Characterization of hyperplastic foci observed in surgical specimens of hepatocellular carcinoma.

By reviewing previous surgical specimens of hepatocellular carcinoma, 17 cases with hyperplastic foci (HPF) characterized by discernible increase in nuclear densities, could be histologically selected. Nuclear densities of HPF and control hepatic parenchyma were assessed quantitatively by counting the nuclear number of hepatic cells, and proliferating cell nuclear antigen labeling index was measured. HPF occurred multifocally, confined within a lobular unit, smoothly merging into surrounding hepatic parenchyma. Nuclear densities of HPF were 1.71 times greater than those of control hepatic parenchyma. The hepatocytes of HPF also showed significantly higher proliferative activities than those of control parenchyma. In addition, noticeable structural distortions, such as focal trabecular thickening or microacinar formation of hepatocytes, were sometimes observed in HPF. However, these HPF seemed to be distinguished from minute de novo hepatocellular carcinoma (HCC) or intrahepatic HCC metastasis, because of paucity of distinctive atypical changes, and intimate correlation with neighboring hepatocytes. Several adjacent HPF were aggregated to form a much larger unit of a hyperplastic area with loss of fibrous septa of liver cirrhosis. It was suggested that grossly detectable large regenerative nodules are produced via fusion of several adjacent HPF.

Enzymatic synthesis of sulfated disaccharides using beta-D-galactosidase-catalyzed transglycosylation.

We have established a unique enzymatic approach for obtaining sulfated disaccharides using Bacillus circulans beta-D-galactosidase-catalyzed 6-sulfo galactosylation. When 4-methyl umbelliferyl 6-sulfo beta-D-galactopyranoside (S6Gal beta-4MU) was used as a donor, the enzyme induced transfer of 6-sulfo galactosyl residue to GlcNAc acceptor. As a result, the desired compound 6'-sulfo N-acetyllactosamine (S6Gal beta1-4GlcNAc) and its positional isomer 6'-sulfo N-acetylisolactosamine (S6Gal beta1-6GlcNAc) were observed by HPAEC-PAD, in 49% total yield based on the donor added, and in a molar ratio of 1:3.5. With a glucose acceptor, the regioselectivity was substantially changed and S6Gal beta1-2Glc was mainly produced along with beta-(1-1)alpha, beta-(1-3), beta-(1-6) isomers in 74% total yield. When methyl alpha-D-glucopyranoside (Glc alpha-OMe) was an acceptor, the enzyme also formed mainly S6Gal beta1-2Glc alpha-OMe with its beta-(1-6)-linked isomer in 41% total yield based on the donor added. In both cases, it led to the predominant formation of beta-(1-2)-linked disaccharides. In contrast, with the corresponding methyl beta-D-glucopyranoside (Glc beta-OMe) acceptor, S6Gal beta1-3Glc beta-OMe and S6Gal beta1-6Glc beta-OMe were formed in a low total yield of 12%. These results indicate that the regioselectivity and efficiency on the beta-D-galactosidase-mediated transfer reaction significantly depend on the anomeric configuration in the glucosyl acceptors.