The multi-functional protein, lactoferrin, a metal-binding glycoprotein with a molecular weight of 80 kDa, belongs to the transferrin family and is found in many secretory fluids and blood. Industrially, bovine lactoferrin is purified from milk or cheese whey and human lactoferrin can be produced by genetic manufacturing. In this special issue, a number of specialists present the most up-to-date information on lactoferrin.

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To favor the extension of human lactoferrin (hLF) and bovine lactoferrin (bLF) assay from research or specialized centers to routine clinical laboratories, we developed a latex agglutination turbidimetric immunoassay (LA) method that is simple to use, yet reliable. Based on the formation of immunocomplexes in the presence of antibody-coated latex particles, it overcomes many of the technical drawbacks show by ELISA methods, such as long incubation times and the need for special equipment and skilled personnel.
This assay also is particularly suited to routine work: the test time is short 10 min (fully automated assay); the measurement range is broad (from ～40 to 1600 ng/ml for hLF and bLF); there is a high correlation to the ELISA method (r=0.9944); and automation is easy. Here we describe characteristics and performance of the methods.

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cDNAs and genomic DNA for lactoferrin from many species, including the human and bovine forms, have been cloned and analyzed. By transgenic technology, it is now possible to produce recombinant lactoferrin in microorganisms such as bacteria, fungi and yeast, in animals such as mice and cows, and in plants such as rice and maize. In particular, transgenic brown rice has been found to produce recombinant human lactoferrin at very high levels of 5g/kg. Such transgenic plants could clearly be used as functional foodstuffs directly or as sources of pharmaceuticals or supplements from the viewpoint of molecular farming.

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Lactoferrin (Lf), a major iron-binding protein in human milk, has been suggested to have multiple biological roles. Mammalian Lf receptors (LfR) are considered to be responsible for mediating some of the functions of Lf. We have cloned a small intestinal LfR (SI-LfR) and elucidated biochemical characteristics, properties of interaction with Lf, and its expression levels in human small intestine and in Caco-2 cells. We then studied the cellular internalization of hLf in differentiated Caco-2 cells grown as monolayers on Transwell inserts, comparing it to that of human transferrin (hTf). We have found that hLf was internalized from only the apical side and localized to nuclei, but hTf was internalized from the basolateral side, not from the apical, and localized in the cytoplasm. As a step towards detailed characterization of the Lf-LfR interaction, we used a series of Lf chimeras to analyze which part of hLf is responsible for the interaction with the SI-LfR in Caco-2 cells. An N-terminus 1-90 amino acid sequence of hLf was found to be sufficient for binding, internalization and targeting to the nucleus. One of the functions of the SI-LfR is SI-LfR-mediated uptake of iron into the small intestine. In Caco-2 cells transfected with the SI-LfR gene, ¹²⁵I-hLf binding and ⁵⁹Fe-hLf uptake were increased by 1.7 and 3.4 times, respectively, compared to mock-transfected cells. One of the functions of the SI-LfR is therefore SI-LfR- mediated uptake of iron into the small intestine. SI-LfR may also mediate other functions of Lf, such as modulating immune function, and enhancing cell proliferation. The detailed mechanisms behind the interaction between SI-LfR and Lf still need to be elucidated.

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At least 6 lactoferrin (LF) -binding proteins (LF-BPs :p15, p17, p25, p27, p35 and p37) have been purified from bLF fractions. They were here investigated for: (i) identity, determined from their partial N-terminal amino acid sequences; (ii) physiological correlations with bLF and regulation through phosphorylation by protein kinases in vitro; and (iii) effects of binding of cholesterol-3-sulfate (CH-3S) or sulfatide to p37 (FGF-BP) on binding to bLF in vitro. In the last case reduction was found. The results and indirect information obtained suggest that bLF may play an physiological role as a carrier for several bLF-binding functional proteins in vivo.

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Starch seems to be a simple material because it contains only glucose. However, we do not yet fully understand even its primary structure. According to many recent studies on the structure, starch has four levels of structures: fine, crystal, blocklet and ultra-structures (granular architecture). We have gathered the major hypotheses and the data regarding starch structure and behavior, and demonstrated comprehensively the blocklet concept, the blocklet proof, blocklet architecture and the blocklet arrangement in the granules in this review. In the architecture of blocklet and granular ultra-structure, the roles of the starch molecules such as amylopectin, amylose and intermediate materials, the non-starch molecules such as bound phosphates and lipids, and the crystal dimensions etc., are different. The amylopectin plays a main role in blocklet architecture, while the other components may be important in contributing to the strength and flexibility of the starch granule. The two types of blocklet, normal and defective blocklets, may arrange into the two formations, heterogeneous shell and homogenous shell from the viewpoint of the physicochemical properties of the starch granules.

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Cholesterol has been implicated in the pathogenesis of Alzheimer’s disease (AD). Although the underlying mechanisms are not yet clear, several studies have provided evidence for the involvement of cholesterol-rich lipid rafts in the production of amyloid β peptide (Aβ), the major component of amyloid deposits in AD. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids, and proteins can be selectively included or excluded from these microdomains. Lipid rafts have been shown to function as a concentrating platform for a variety of signal transduction molecules. Aβ is generated by two sequential proteolytic cleavage steps from the β-amyloid precursor protein (βAPP). βAPP is initially cleaved by β-secretase, which has been identified as a β-site APP-cleaving enzyme (BACE), followed by the subsequent intramembrane proteolysis of the membrane-bound C-terminal fragment (βCTF, C99) catalyzed by γ-secretase. Recent reports have suggested that β- and γ-secretase are localized in lipid rafts and cholesterol can regulate these activities or distributions. These data demonstrated that lipid rafts offer a structural platform for examining cholesterol’s effect on Aβ generation. The association of the Aβ-producing enzyme with lipid rafts and its relation to cholesterol metabolism will provide new insights for understanding the mechanism of Aβ generation in AD.

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L-Ascorbic acid (AsA) has diverse physiological activities and is widely used as a nutritional supplement. However, it is disadvantageous that AsA is easily degraded under various oxidative conditions. To solve this problem, we developed a stable derivative of AsA, L-ascorbic acid 2-glucoside (AA-2G), which was synthesized by regioselective transglycosylation with cyclodextrin glucanotransferase from Bacillus stearothermophilus. AA-2G was very stable in neutral solution and resistant to heat, light and oxidation. This vitamin derivative was verified to release AsA by hydrolysis with α-glucosidase and to exhibit its physiological activities in vivo. Moreover, it was confirmed that AA-2G was little affected by coexisting materials such as metallic ions, proteins and vitamins and was highly stable in various food products. We also investigated the metabolic fate of AA-2G following ingestion. As a result, ingested AA-2G was clarified to behave as AsA in the body after the hydrolysis by intestinal brush border α-glucosidase. In addition, various toxicological tests showed that AA-2G was a highly safe material. From these findings, AA-2G is considered to be available effectively as a stable vitamin C supplement in the field of foods.

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Seven circumstances or mechanisms are indicated whereby fibre and polyols could reduce blood glucose and insulin concentrations and so potentially help to reduce the prevalence of diabetes, CHD, and certain cancers. All are independent of possible additional influences by associated substances such as antioxidant lignans. Further, high cereal fibre intake additionally lowers the starch:fibre ratio thus lowering the glycaemic index of total carbohydrate while not affecting that of available carbohydrate; this may be helped by fibre in whole grain causing some starch to be unavailable. It seems that glycaemic index expressed as g equivalents per g available carbohydrate does not account for the full effects of fibre on lowering of blood glucose in some circumstances whereas glycaemic load in units of g equivalents per day (or per unit food) would. Overall, the glycaemic load appears stronger at present than glycaemic index for: the epidemiology on development of type-2 diabetes and heart disease combined; the epidemiology of HDL, TG, and C-reactive protein; the interventions on body weight in ad-libitum studies; and the interventions on glycated proteins and fasting blood glucose in diabetics. These data appear more consistent than is generally discussed in the literature, and indicates a need for a more analytical assessment of the evidence base than undertaken so far by either national authorities or associations of health professionals or by application of simple fixed-effects meta-analyses. Scope exists for a reduction in glycaemic load by replacing high glycaemic available carbohydrate with either low-glycaemic available carbohydrate or fibre or polyols and this potentially could reduce the prevalence of diabetes and CHD by up to thirty percent.

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