Determination of birefringence and slow axis distribution using an interferometric measurement system with liquid crystal phase shifter.

It is known that liquid crystal (LC) cells are useful as compact and easy-to-handle phase shifters that are readily coupled into the optics of standard microscope systems. Here, a uniformly aligned molecular LC phase shifter is introduced into a polarization microscope to attain a birefringence imaging system, using the phase-shift interferometric technique. Since the birefringence can be determined accurately only when the optical axis of the sample is parallel or perpendicular to the slow axis (variable axis) of the LC phase shifter, an improved data analysis method is proposed for determining the birefringence independently of the direction; a simple method of determining the slow axis distribution is also demonstrated. Measurements of the birefringence and slow axis distribution properties of a potato starch particle are demonstrated to confirm the novel determination method.

A crustacean Ca2+-binding protein with a glutamate-rich sequence promotes CaCO3 crystallization.

The DD4 mRNA of the penaeid prawn Penaeus japonicus was shown previously to be expressed in the epidermis adjacent to the exoskeleton specifically during the post-moult period, when calcification of the exoskeleton took place. The encoded protein possessed a Ca2+-binding site, suggesting its involvement in the calcification of the exoskeleton. In the present study, an additional ORF (open reading frame) of 289 amino acids was identified at the 5' end of the previous ORF. The newly identified part of the encoded protein included a region of approx. 120 amino acids that was highly rich in glutamate residues, and contained one or more Ca2+-binding sites. In an immunohistochemical study, signals were detected within calcified regions in the endocuticular layer of the exoskeleton. Bacterially expressed partial segments of the protein induced CaCO3 crystallization in vitro. Finally, a reverse transcription-PCR study showed that the expression was limited to an early part of the post-moult period, preceding significant calcification of the exoskeleton. These observations argue for the possibility that the encoded protein, renamed crustocalcin (CCN), promotes formation of CaCO3 crystals in the exoskeleton by inducing nucleation.

Characterization of Prismalin-14, a novel matrix protein from the prismatic layer of the Japanese pearl oyster (Pinctada fucata).

The mollusc shell is a hard tissue consisting of calcium carbonate and organic matrices. The organic matrices are believed to play important roles in shell formation. In the present study, we extracted and purified a novel matrix protein, named Prismalin-14, from the acid-insoluble fraction of the prismatic layer of the shell of the Japanese pearl oyster (Pinctada fucata), and determined its whole amino acid sequence by a combination of amino acid sequence analysis and MS analysis of the intact protein and its enzymic digests. Prismalin-14 consisted of 105 amino acid residues, including PIYR repeats, a Gly/Tyr-rich region and N- and C-terminal Asp-rich regions. Prismalin-14 showed inhibitory activity on calcium carbonate precipitation and calcium-binding activity in vitro. The scanning electron microscopy images revealed that Prismalin-14 affected the crystallization of calcium carbonate in vitro. A cDNA encoding Prismalin-14 was cloned and its expression was analysed. The amino acid sequence deduced from the nucleotide sequence of Prismalin-14 cDNA was identical with that determined by peptide sequencing. Northern-blot analysis showed that a Prismalin-14 mRNA was expressed only at the mantle edge. In situ hybridization demonstrated that a Prismalin-14 mRNA was expressed strongly in the inner side of the outer fold of the mantle. These results suggest that Prismalin-14 is a framework protein that plays an important role in the regulation of calcification of the prismatic layer of the shell.

A novel calcium-binding peptide from the cuticle of the crayfish, Procambarus clarkii.

A novel peptide named calcification-associated peptide (CAP)-2 was isolated from the exoskeleton of the crayfish, Procambarus clarkii. CAP-2 consists of 65 amino acid residues and has a 44% sequence identity with CAP-1 characterized previously. It has a chitin-binding domain observed in many arthropod cuticle proteins. CAP-2 showed inhibitory activity on calcium carbonate precipitation and chitin-binding ability. A CAP-2 cDNA was cloned using RT-PCR and RACE and the open reading frame encoded a precursor peptide consisting of a signal peptide and CAP-2. RT-PCR revealed that CAP-2 mRNA was exclusively expressed in the epidermal tissue during the postmolt stage, the site and stage being associated with calcification. Calcium-binding assay using recombinant CAP-2 revealed that this peptide had affinity for calcium ions with a Kd value of about 1 mM. All these results suggest that CAP-2 serves as a nucleator or a regulator in the calcification of the exoskeleton.

Cloning and expression of a cDNA encoding a matrix peptide associated with calcification in the exoskeleton of the crayfish.

Calcification-associated peptide (CAP)-1 isolated from the exoskeleton of the crayfish, Procambarus clarkii, has anti-calcification activity and chitin-binding ability and is, therefore, considered to be associated with calcification. In this study, a cDNA encoding CAP-1 was cloned and characterized. An open reading frame encoded a pre-propeptide of 99 amino acid residues, which was composed of a signal peptide, a CAP-1 precursor and two-basic amino acid residues at the C-terminus. The dibasic residues were not observed in the natural CAP-1. Expression analyses using Northern blot and RT-PCR revealed that the mRNA encoding CAP-1 was strongly expressed in the epidermal tissue during the postmolt stage, where and when the calcification takes place. These results support that CAP-1 may play an important role in the calcification of the exoskeleton. Based on the nucleotide sequence of the cDNA encoding CAP-1, a recombinant CAP-1 and that carrying the basic residues at the C-terminus were expressed in Escherichia coli. Anti-calcification assay showed that these recombinant peptides were less active than natural CAP-1, indicating that the phosphate group at the 70th residue, Ser, in natural CAP-1 is important for inhibitory activity and that the paired basic residues have some contribution to the elevation of inhibitory activity.