Identification of μ-, m-calpains and calpastatin and capture of μ-calpain activation in endothelial cells

The presence of the calpain-calpastatin system in human umbilical vein endothelial cells (HUVEC) was investigated by means of ion exchange chromatography, Western blot analysis, and Northern blot analysis. On DEAE anion exchange chromatography, calpain and calpastatin activities were eluted at approximately 0.30 M and 0.15-0.25 M NaCl, respectively. For half-maximal activity, the protease required 800 μM Ca²⁺, comparable to the Ca²⁺ requirement of m-calpain. By Western blot analysis, the large subunit of μ-calpain (80 kDa) was found to be eluted with calpastatin (110 kDa). Both the large subunit of m-calpain (80 kDa) and calpastatin were detected in the respective active fractions. By Northern blot analysis, mRNAs for large subunits of μ- and m-calpains were detected in single bands, each corresponding to approximately 3.5 Kb. Calpastatin mRNA was observed in two bands corresponding to approximately 3.8 and 2.6 Kb. Furthermore, the activation of μ-calpain in HUVEC by a calcium ionophore was examined, using an antibody specifically recognizing an autolytic intermediate form of μ-calpain large subunit (78 kDa). Both talin and filamin of HUVEC were proteolyzed in a calcium-dependent manner, and the reactions were inhibited by calpeptin, a cell-permeable calpain specific inhibitor. Proteolysis of the cytoskeleton was preceded by the appearance of the autolytic intermediate form of μ-calpain, while the fully autolyzed postautolysis form of μ-calpain (76 kDa) remained below detectable levels at all time points examined. These results indicate that the calpain-calpastatin system is present in human endothelial cells and that μ-calpain may be involved in endothelial cell function mediated by Ca²⁺ via the limited proteolysis of various proteins. J. Cell. Biochem. 66:197-209, 1997. © 1997 Wiley-Liss, Inc.     

Cutaneous Photobiology. The Melanocyte vs. the Sun: Who Will Win the Final Round?

Solar ultraviolet radiation (UV) is a major environmental factor that dramatically alters the homeostasis of the skin as an organ by affecting the survival, proliferation and differentiation of various cutaneous cell types. The effects of UV on the skin include direct damage to DNA, apoptosis, growth arrest, and stimulation of melanogenesis. Long‐term effects of UV include photoaging and photocarcinogenesis. Epidermal melanocytes synthesize two main types of melanin: eumelanin and pheomelanin. Melanin, particularly eumelanin, represents the major photoprotective mechanism in the skin. Melanin limits the extent of UV penetration through the epidermal layers, and scavenges reactive oxygen radicals that may lead to oxidative DNA damage. The extent of UV‐induced DNA damage and the incidence of skin cancer are inversely correlated with total melanin content of the skin. Given the importance of the melanocyte in guarding against the adverse effects of UV and the fact that the melanocyte has a low self‐renewal capacity, it is critical to maintain its survival and genomic integrity in order to prevent malignant transformation to melanoma, the most fatal form of skin cancer. Melanocyte transformation to melanoma involves the activation of certain oncogenes and the inactivation of specific tumor suppressor genes. This review summarizes the current state of knowledge about the role of melanin and the melanocyte in photoprotection, the responses of melanocytes to UV, the signaling pathways that mediate the biological effects of UV on melanocytes, and the most common genetic alterations that lead to melanoma.     

The histological analysis,colorimetric evaluation,and chemical quantification of melanin content in ‘suntanned’ fish

Human melanocytes respond to UV irradiation by increasing the synthesis of melanin. While much is now understood of the pathways governing this process and the nature of the melanin synthesized, little is known of melanins produced by lower vertebrates and their capacity to respond to UV. Here we report that a fish, red seabream, can undergo ‘suntanning’. Histological, colorimetric and chemical assays were performed for suntanned red seabream fish bred in net cages to analyse the melanins and compared with shaded or wild red seabream fish. For color evaluation, the L* values of suntanned fish were dramatically lower than those in the other two groups. Pyrrole‐2,3,5‐tricarboxylic acid (PTCA), an indicator of eumelanin, was detected in suntanned fish at five times higher levels than in shaded or wild fish while 4‐amino‐3‐hydroxyphenyl‐alanine (4‐AHP), a marker for pheomelanin, could not be detected in any of the samples. Histological analysis showed that melanocytes in the suntanned skin enlarged and increased in number to form a monolayer at the surface of the skin. Analysis of L* values and PTCA levels showed quite a high correlation coefficient (r = ?0.843). When comparing shaded and wild red seabream fish, the scores were closer but some significant differences were still found in some body areas. These results indicate that eumelanin accumulates in suntanned fish during the increase in skin color, which is induced by sunlight, presumably by ultraviolet radiation.     

Molecular structure and properties of lectin from tomato fruit

A cDNA encoding tomato fruit lectin was cloned from an unripe cherry-tomato fruit cDNA library. The isolated lectin cDNA contained an open reading frame encoding 365 amino acids, including peptides that were sequenced. The deduced sequence consisted of three distinct domains: (i) an N-terminal short extensin-like domain; (ii) a Cys-rich carbohydrate binding domain composed of four almost identical chitin-binding domains; (iii) an internal extensin-like domain of 101 residues containing 15 SerPro(4) motifs inserted between the first and second chitin-binding domains. The molecular weight of the lectin was 65,633 and that of the deglycosylated lectin was 32,948, as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This correlated with the estimated molecular weight of the deduced sequence. Recombinant tomato lectin expressed in Pichia pastoris possessed chitin-binding but not hemagglutinating activity. These findings confirmed that the cDNA encoded tomato lectin.     

Quantitative Analysis of Eumelanin and Pheomelanin in Humans,Mice, and Other Animals: a Comparative Review

The color of hair, skin, and eyes in animals mainly depends on the quantity, quality, and distribution of the pigment melanin, which occurs in two types: black to brown eumelanin and yellow to reddish pheomelanin. Microanalytical methods to quantify the amounts of eumelanin and pheomelanin in biological materials were developed in 1985. The methods are based on the chemical degradation of eumelanin to pyrrole‐2,3,5‐tricarboxylic acid and of pheomelanin to aminohydroxyphenylalanine isomers, which can be analyzed and quantitated by high performance liquid chromatography. This review summarizes and compares eumelanin and pheomelanin contents in various pigmented tissues obtained from humans, mice, and other animals. These methods have become valuable tools to study the functions of melanin, the control of melanogenesis, and the actions and interactions of pigmentation genes. The methods have also found applications in many clinical studies. High levels of pheomelanin are found only in yellow to red hairs of mammals and in red feathers of birds. It remains an intriguing question why lower vertebrates such as fishes do not synthesize pheomelanin. Detectable levels of pheomelanin are detected in human skin regardless of race, color, and skin type. However, eumelanin is always the major constituent of epidermal melanin, and the skin color appears to be determined by the quantity of melanin produced but not by the quality.     

Improvement of bacterial transformation efficiency using plasmid artificial modification

We have developed a method to improve the transformation efficiency in genome-sequenced bacteria, using ‘Plasmid Artificial Modification’ (PAM), using the host''s own restriction system. In this method, a shuttle vector was pre-methylated in Escherichia coli cells, which carry all the putative genes encoding the DNA modification enzymes of the target microorganism, before electroporation was performed. In the case of Bifidobacterium adolescentis ATCC15703 and pKKT427 (3.9 kb E. coli-Bifidobacterium shuttle vector), introducing two Type II DNA methyltransferase genes lead to an enhancement in the transformation efficiency by five orders of magnitude. This concept was also applicable to a Type I restriction system. In the case of Lactococcus lactis IO-1, by using PAM with a putative Type I methyltransferase system, hsdMS1, the transformation efficiency was improved by a factor of seven over that without PAM.     

Systematic approaches to using the FOX hunting system to identify useful rice genes

Ectopic gene expression, or the gain-of-function approach, has the advantage that once the function of a gene is known the gene can be transferred to many different plants by transformation. We previously reported a method, called FOX hunting, that involves ectopic expression of Arabidopsis full-length cDNAs in Arabidopsis to systematically generate gain-of-function mutants. This technology is most beneficial for generating a heterologous gene resource for analysis of useful plant gene functions. As an initial model we generated more than 23 000 independent Arabidopsis transgenic lines that expressed rice fl-cDNAs (Rice FOX Arabidopsis lines). The short generation time and rapid and efficient transformation frequency of Arabidopsis enabled the functions of the rice genes to be analyzed rapidly. We screened rice FOX Arabidopsis lines for alterations in morphology, photosynthesis, element accumulation, pigment accumulation, hormone profiles, secondary metabolites, pathogen resistance, salt tolerance, UV signaling, high light tolerance, and heat stress tolerance. Some of the mutant phenotypes displayed by rice FOX Arabidopsis lines resulted from the expression of rice genes that had no homologs in Arabidopsis . This result demonstrated that rice fl-cDNAs could be used to introduce new gene functions in Arabidopsis. Furthermore, these findings showed that rice gene function could be analyzed by employing Arabidopsis as a heterologous host. This technology provides a framework for the analysis of plant gene function in a heterologous host and of plant improvement by using heterologous gene resources.     

NMR‐based characterization of a refolding intermediate of β2‐microglobulin labeled using a wheat germ cell‐free system

In patients with dialysis‐related amyloidosis, β2‐microglobulin (β2‐m) is a major structural component of amyloid fibrils. It has been suggested that the partial unfolding of β2‐m is a prerequisite to the formation of amyloid fibrils, and that the folding intermediate trapped by the non‐native trans‐Pro32 isomer leads to the formation of amyloid fibrils. Although clarifying the structure of this refolding intermediate by high resolution NMR spectroscopy is important, this has been made difficult by the limited lifetime of the intermediate. Here, we studied the structure of the refolding intermediate using a combination of amino acid selective labeling with wheat germ cell‐free protein synthesis and NMR techniques. The HSQC spectra of β2‐ms labeled selectively at either phenylalanine, leucine, or valine enabled us to monitor the structures of the refolding intermediate. The results suggested that the refolding intermediate has an overall fold and cores similar to the native structure, but contains disordered structures around Pro32. The fluctuation of the β‐sheet regions especially the last half of the βB strand and the first half of the βE strand, both suggested to be important for amyloidogenicity, may transform β2‐m into an amyloidogenic structure.     

MHC class I A loci polymorphism and diversity in three Southeast Asian populations of cynomolgus macaque

Cynomolgus macaques (Macaca fascicularis, Mafa) have emerged as important animal models for biomedical research, necessitating a more extensive characterization of their major histocompatibility complex polymorphic regions. The current information on the polymorphism or diversity of the polygenetic Mafa class I A loci is limited in comparison to the more commonly studied rhesus macaque Mafa class I A loci. Therefore, in this paper, to better elucidate the degree and types of polymorphisms and genetic differences of Mafa-A1 among three native Southeast Asian populations (Indonesian, Vietnamese, and Filipino) and to investigate how the allele differences between macaques and humans might have evolved to affect their respective immune responses, we identified 83 Mafa-A loci-derived alleles by DNA sequencing of which 66 are newly described. Most alleles are unique to each population, but seven of the most frequent alleles were identical in sequence to some alleles in other macaque species. We also revealed (1) the large and dynamic genetic and structural differences and similarities in allelic variation by analyzing the population allele frequencies, Hardy-Weinberg’s equilibrium, heterozygosity, nucleotide diversity profiles, and phylogeny, (2) the difference in genetic structure of populations by Wright’s FST statistic and hierarchical analysis of molecular variance, and (3) the different demographic and selection pressures on the three populations by performing Tajima’s D test of neutrality. The large level of diversity and polymorphism at the Mafa-A1 was less evident in the Filipino than in the Vietnam or the Indonesian populations, which may have important implications in animal capture, selection, and breeding for medical research.