Vascular endothelial growth factor promotes cell-cycle transition from G0 to G1 phase in subcultured endothelial cells of diabetic rat thoracic aorta.

Vascular endothelial growth factor (VEGF) has been claimed to be a major positive regulator of angiogenesis in diabetic retinopathy and atherosclerosis. Diabetic state-induced alteration of the phenotypes and the influence of 12-h pretreatment with VEGF were examined after a further 12-h treatment with only 1% fetal bovine serum in subcultured endothelial cells (EC) derived from rat thoracic aorta. By flow cytometric cell cycle analysis, VEGF showed quite different transition patterns from those of platelet-derived growth factor (PDGF) in 5-day (at the progression phase) cultured normal rat EC even though VEGF belongs to the PDGF family. VEGF promoted cell cycle transition from the G0 to the G1 phase at 3 ng/ml, but at 30 ng/ml, VEGF weakly inhibited it compared with the effect of PDGF. The streptozotocin-diabetic state promoted cell cycle transition of EC from the G0 to the G1 phase. The promotion by the low concentration of VEGF was observed even at the point of 35-day culture (angiogenic EC at the competence phase in normal state). The diabetic state enhanced EC proliferation rather than tube formation, and the tube formation was scarce. The promotion of cell cycle transition by VEGF may aggravate furthermore diabetic angiopathy due to the leaky constitution of blood vessels.

One Step Synthesis of Inverted Aspartame Type Sweetener, Ac-Phe-Lys, Using Chemically Modified Chymotrypsin.

To search for techniques of simplified peptide synthesis, benzyloxycarbonyl chymotrypsin was prepared by a water-soluble acylating reagent and used to make Ac-Phe-Lys, an artificial peptide sweetener, which was selected as a target compound. As a result of using chemically modified chymotrypsin, Lys can be coupled directly with Ac-Phe and Ac-Phe-Lys made virtually in one step. Moreover, the total yield from preparation and purification steps for Ac-Phe-Lys was 13%. The value corresponds to that of the chemical synthesis method. On the contrary, enzymatic synthesis using native chymotrypsin cannot reach the level of the new method. It is expected that the method is more effective for simplified peptide synthesis as compared with other methods, especially on a large scale.

Changes of amino acid levels and aspartate distribution in the cervical spinal cord after traumatic spinal cord injury.

To evaluate the role of excitatory amino acids in secondary injury occurring after spinal cord trauma, several experimental studies focusing on the the changes of amino acid levels in the spinal cord have been performed to date. However, because of technical limitations, it has not been possible to correlate the local changes of excitatory amino acids with the total tissue levels of excitatory amino acids. To investigate the connection between the spread of injury and the excitatory amino acids, we assessed, the local changes of aspartate through novel experimental approaches like immunoreactivity via fluorescence microphotometry and histopathology while also analyzing the total tissue levels of amino acids via HPLC. These studies were performed using a model of incomplete cervical spinal cord injury in rats. Through this approach, we found that the levels of excitatory amino acids, such as glutamate and aspartate, began to decrease immediately after injury. No significant decrease was observed in the other amino acids. Similarly, local changes in aspartate in the spinal cord were observed using fluorescence microphotometry. The decrease in the anterior and posterior horns was rapid up to 15 min after injury, but, slowed thereafter, suggesting that a release of excitatory amino acids occurred at the site of primary injury almost immediately following injury. At 15-min post-injury large neurons within the injured cord appeared intact on histopathological analysis demonstrating that the alteration of excitatory amino acids occurs prior to histopathological change. Histopathological change in the white matter occurred more slowly than in the anterior and posterior horns, suggesting the spread of the lesion by secondary damage due to an autoclastic mechanism.

Tastes produced by peptides containing ionic groups and by related compounds.

The typical tastes imparted by ionic groups are salty-like of sodium chloride and umami-like of monosodium glutamate, but the relationship between the taste and chemical structure has not previously been elucidated. One of the reason for the difficulty in understanding the taste-structure relationship is the presence of the ambiguous and unfavorable tastes of neutral salts. We define the strange tastes of neutral salts (TNS) collectively in a specific category, and then the tastes due to ionic groups. Sour, salty, and umami tastes and TNS were studied for different acidic and basic groups and various combinations of both groups to elucidate the taste characteristics of the ionic groups. The results reveal that the tastes due to ionic groups have common characteristics, being different from bitter and sweet tastes.

Role of basic and acidic fragments in delicious peptides (Lys-Gly-Asp-Glu-Glu-Ser-Leu-Ala) and the taste behavior of sodium and potassium salts in acidic oligopeptides.

The role of the acidic fragment (Asp-Glu-Glu) in delicious peptides was investigated in detail by using the Na+ or K+ salts of acidic oligopeptides so that amount of Na+ or K+ intake of peptides composed of acidic amino acids could be varied by changing their sequences. The taste of these peptides was confirmed to vary with Na+ or K+ intake. Additionally, in order to study the role of basic (Lys-Gly) and acidic (Asp-Glu-Glu) fragments in delicious peptides for producing the taste, five delicious peptide analogs, Ser-Leu-Ala-Lys-Gly-Asp-Glu-Glu, Ser-Leu-Ala-Asp-Glu-Glu-Lys-Gly, Lys-Gly-Ser-Leu-Ala-Asp-Glu-Glu, Lys-Gly-Asp-Glu-Glu, and Glu-Glu-Asp-Gly-Lys, were synthesized. The intensity of the umami and/or salty taste of these peptides and their Na salts was almost the same, despite their chemical structures being different. These results indicate that the acidic fragment as well as the basic fragment plays an important role in the taste production and intensity of delicious peptides, and that an umami or salty taste can be produced by the localization of the cation of the basic fragment and the anion of the acidic fragment.

Relationship between Taste and Structure of O-Aminoacyl Sugars Containing Basic Amino Acids.

To study the relationship between taste and structure of O-aminoacyl sugars, a number of O-aminoacyl sugars containing basic amino acids were prepared. Taste quality of O-aminoacyl sugars was dependent on the side chain length of basic amino acids that were introduced into sugars. O-Aminoacyl sugars had an excellent sodium ion diet effect that could reduce sodium ion intake to 10%.

Characteristics of chymotrypsin modified with water-soluble acylating reagents and its peptide synthesis ability in aqueous organic media.

Several kinds of modified chymotrypsin were prepared with water-soluble acylating reagents, and their characteristics after hydrolyzing with unmodified chymotrypsin in aqueous-N,N'-dimethylformamide (DMF) media were compared. It was found that chymotrypsin (Csin), of which a 20% amino group was modified with a benzyloxycarbonyl group (Z(20)Csin), had more favorable characteristics than unmodified chymotrypsin with regard to hydrolytic activity in an aqueous DMF media. We also investigated the Z(20)Csin-catalyzed peptide synthesis in two different solution systems. In the one-layer system containing water and DMF, Z(20)Csin catalyzed the peptide bond formation in a higher yield than that by unmodifide chymotrypsin and enabled a synthetic reaction in even an 80% (v/v) DMF media, in which the hydrolytic reaction could not be carried out. Z(20)Csin catalyzed the condensation between some N-acyl amino acids or peptide derivatives and amino acids in 90% ethylacetate, 90% hexane or 50% benzene. This latter method employs a two-layer system, and the modified enzyme may be able to reduce the number of synthetic steps when preparing acyl peptides.

Mechanism for the bitter tasting potency of peptides using O-aminoacyl sugars as model compounds.

In order to study the role of hydrophobicity in bitter peptides, several O-aminoacyl sugars, in which amino acids or peptides were attached to the 2- and 3-position of methyl alpha-D-glucopyranoside, were synthesized and sensory analyses were carried out. It was found that the bitterness increased as the hydrophobicity of compounds increased, implying that the bitterness receptor recognizes the hydrophobicity of bitter peptides. A structure for the bitterness receptor is also discussed.

Practical debittering using model peptides and related compounds.

In order to develop a practical debittering method for amino acids and peptides, several debittering methods were studied. The authors found that hooking acidic amino acids to and acetylation of bitter amino acids is very effective to remove the bitterness from their concentrated solution. For debittering by mixing with additives, skin milk and other peptide compounds were effective. Acidic amino acids were also effective to reduce the bitterness. Gelatinized starch was found to be useful for debittering because it takes bitter substances into its net structure.