Multiple origins for phenylketonuria in Europe

Phenylketonuria (PKU), a disorder of amino acid metabolism prevalent among Caucasians and other ethnic groups, is caused primarily by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). PKU is a highly heterogeneous disorder, with more than 60 molecular lesions identified in the PAH gene. The haplotype associations, relative frequencies, and distributions of five prevalent PAH mutations (R158Q, R261Q, IVS10nt546, R408W, and IVS12n1) were established in a comprehensive European sample population and subsequently were examined to determine the potential roles of several genetic mechanisms in explaining the present distribution of the major PKU alleles. Each of these five mutations was strongly associated with only one of the more than 70 chromosomal haplotypes defined by eight RFLPs in or near the PAH gene. These findings suggest that each of these mutations arose through a single founding event that occurred within time periods ranging from several hundred to several thousand years ago. From the significant differences observed in the relative frequencies and distributions of these five alleles throughout Europe, four of these putative founding events could be localized to specific ethnic subgroups. Together, these data suggest that there were multiple, geographically and ethnically distinct origins for PKU within the European population.     

Using microparticle labeling and counting for attomole-level detection in heterogeneous immunoassay.

A new heterogeneous "sandwich" immunoassay utilizing microparticles as labels to realize high sensitivity is described. In this method, antibody fixed on the microparticles reacts with antigen previously trapped on a microplate surface, which makes the antigen molecules visible and countable with an inverted optical microscope. The method is highly sensitive because the reacted single microparticle, therefore single antigen molecule, can be detected. The sensitivity depends both on the reaction efficiency of the immunoreaction and on nonspecific adsorption of the microparticles on the microplate surface. Therefore, the protocol for preparing microparticle having antibody on the surface and a microplate having capture antibody was investigated to realize high sensitivity. Carboxylated microparticles of 0.76 microns in diameter were conjugated with affinity-purified antibody using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. It was determined that 1 g microparticles had 880 micrograms antibody (approximately 1100 antibody molecules per 1 microparticle). The immunoreaction efficiency reached 18% at 1 x 10(-13) mol/liter antigen concentration. The lower detection limit was 3.1 x 10(-14) mol/liter (1.6 amol) using human alpha-fetoprotein as a model antigen.     

An Integrated Map with Cosmid/PAC Contigs of a 4-Mb Down Syndrome Critical Region

The major phenotypic features of Down syndrome have been correlated with partial trisomies of chromosome 21, allowing us to define the candidate gene region to a 4-Mb segment on the 21q22.2 band. We present here a high-resolution physical map with megabase-sized cosmid/PAC contigs. This ordered clone library has provided unique material for the integration of a variety of mappable objects, including exons, cDNAs, restriction sites, etc. Furthermore, our results have exemplified a strategy for the completion of the chromosome 21 map to sequencing.     

Phytochrome inhibits the effectiveness of gibberellins to induce cell elongation in rice

Red light controls cell elongation in seedlings of rice (Oryza sativa L.) in a far-red-reversible manner (Nick and Furuya, 1993, Plant Growth Regul. 12, 195–206). The role of gibberellins and microtubules in the transduction of this response was investigated in the rice cultivars Nihon Masari (japonica type) and Kasarath (indica type). The dose dependence of mesocotyl elongation on applied gibberellic acid (GA₃) was shifted by red light, and this shift was reversed by far-red light. In contrast, coleoptile elongation was found to be independent of exogenous GA₃. Nevertheless, it was inhibited by red light, and this inhibition was reversed by far-red light. The content of the active gibberellin species GA₁ and GA₄ was estimated by radio-immunoassay. In the mesocotyl, the gibberellin content per cell was found to increase after irradiation with red light, and this increase was far-red reversible. Conversely, the cellular gibberellin content in japonica-type coleoptiles did not exhibit any significant light response. Microtubules reoriented from transverse to longitudinal arrays in response to red light and this reorientation could be reversed by subsequent far-red light in both the coleoptile and the mesocotyl. This movement was accompanied by changes in cell-wall birefringence, indicating parallel reorientations of cellulose deposition. The data indicate that phytochrome regulates the sensitivity of the tissue towards gibberellins, that gibberellin synthesis is controlled in a negative-feedback loop dependent on gibberellin effectiveness, and that at least two hormone-triggered signal chains are linked to the cytoskeleton in rice.Abbreviations D darkness - FR far-red light - GA₃ gibberellic acid - GC-SIM gas chromatography-selected ion monitoring - R red light This work was supported by a grant of the Human Frontier Science Organization to P.N. Advice and organizational support by Prof. M. Furuya (Hitachi Advanced Research Laboratory, Hatoyama, Japan) and Prof. N. Murofushi (Department of Agricultural Chemistry, University of Tokyo, Japan) is gratefully acknowledged. Seeds of both rice cultivars were kindly provided by Dr. O. Yatou (Institute for Radiation Breeding, Hitachi-Ohmiya, Japan), and the antiGA₁ Me-antiserum for the radio-immunoassays by Dr. I. Yamaguchi (Department of Agricultural Chemistry, University of Tokyo, Japan).     

The second phantom aquatic leaf beetle in Japan: Macroplea mutica rediscovery in the wetlands (Coleoptera: Chrysomelidae)

Wetland biodiversity is currently declining on a global scale. Wetland biodiversity understanding is critical for determining the wetlands' conservation value. In this study, Macroplea Samouelle, 1819 (Coleoptera: Chrysomelidae) was discovered in Aomori Prefecture, Honshu Island, Japan. Only two Macroplea species have been recorded in Japan, M. japana (Jacoby, 1885) and M. mutica (Fabricius, 1792). Macroplea japana had been unrecorded for 60 years before being rediscovered in Honshu Island in 2022, and a single adult M. mutica female was discovered in Hokkaido Prefecture in 2003. The discovered individuals were concluded to be M. mutica based on morphological and molecular analyses. Although morphological differences were observed with the Eurasian M. mutica individuals, the male genitalia was nearly identical to M. mutica. For the molecular phylogenetic analysis based on COI and 28S sequences, Macroplea individuals in Japan were clustered with M. mutica on the Eurasian Continent. This is the first record of this species on Honshu Island (and the second in Japan), as well as the first record of adult males. This species would require conservation policies and additional distributional surveys.     

Controlled release of adeno-associated virus from alginate hydrogel microbeads with enhanced sensitivity to ultrasound

Adeno-associated virus (AAV)-based gene therapy holds promise as a fundamental treatment for genetic disorders. For clinical applications, it is necessary to control AAV release timing to avoid an immune response to AAV. Here we propose an ultrasound (US)-triggered on-demand AAV release system using alginate hydrogel microbeads (AHMs) with a release enhancer. By using a centrifuge-based microdroplet shooting device, the AHMs encapsulating AAV with tungsten microparticles (W-MPs) are fabricated. Since W-MPs work as release enhancers, the AHMs have high sensitivity to the US with localized variation in acoustic impedance for improving the release of AAV. Furthermore, AHMs were coated with poly-l -lysine (PLL) to adjust the release of AAV. By applying US to the AAV encapsulating AHMs with W-MPs, the AAV was released on demand, and gene transfection to cells by AAV was confirmed without loss of AAV activity. This proposed US-triggered AAV release system expands methodological possibilities in gene therapy.     

Control by Light of Hypocotyl Elongation and Levels of Endogenous Gibberellins in Seedlings of Lactuca sativa L.

Four 13-hydroxygibberellins, gibberellin A₁ (GA₁), 3-epi-GA₁,GA₁₉ and GA₂₀ were identified by full-scan GC/MS in extractsof lettuce seedlings (Lactuca sativa L. cv. Grand Rapids). Theresults suggest that the early-13-hydroxylation biosyntheticpathway to GA₁ functions in the lettuce seedlings. It was alsofound that GA₁ is active per se in the control of hypocotylelongation in lettuce seedlings. To investigate the relationshipbetween control by light of hypocotyl elongation and levelsof endogenous GAs in lettuce, endogenous levels of GAs werequantified by radioimmunoassay in seedlings that had been grownfor 5 days in the dark (5D) and in those that had been grownfor 4 days in the dark and then under white light for 1 day(4D1L). The endogenous level of GA₁ in the upper and lower partsof hypocotyls in 5D seedlings was about four times higher thanthat in 4D1L seedlings. The response of explants (hypocotylsegments with cotyledons) from dark-grown seedlings to GA₁ isknown to be similar in the dark and under white light when theexplants are treated with inhibitors of the biosynthesis ofGA. Therefore, the above information suggests that the highlevel of GA₁ in hypocotyls of dark-grown seedlings is responsiblefor the rapid elongation of hypocotyl, while irradiation bywhite light decreases the endogenous level of GA₁ in the hypocotylswith a resultant decrease in the rate of hypocotyl elongation. (Received March 13, 1992; Accepted May 21, 1992)     

A 19-kb CpG Island Associated with Single-minded Gene 2 in Down Syndrome Chromosomal Region

To help in isolating the genes involved in Down syndrome, wesought CpG islands in 4 Mb cosmid/PAC contigs spanning mostof the 21q.22.2 band using seven rare cutting enzymes. A strikingfeature was observed upstream of hSIM2 where at least 41 rare-cuttingsites were clustered within a 20-kb region. To investigate thestructure of the cluster, a cosmid containing hSIM2 was submittedto shotgun sequencing. Sequence analysis revealed that the clusterwas a long CpG island extending 19, 128 nucleotides which includesin the first and second exons of hSIM2. Taken together withour observation in which the CpG islands were concentrated within1.2 Mb around hSIM2, we propose that this region functions asan R-band, and the cluster provides a unique element for markingof DNA for the spatial and temporal expression of the hSIM2locus.     

Changes in intracellular location of DNA polymerase-α during oocyte maturation of the toad,bufo bufo japonicus

Intracellular location of DNA polymerase-α during oocyte maturation of the toad was studied. Quantitative and qualitative changes in the activity of DNA polymerase-α were not observed during the maturational process. Nearly all activity was found in isolated germinal vesicles from full grown oocytes and in enucleated mature oocytes. The cytoplasmic DNA polymerase-α of mature oocytes was recovered at buoyant densities equivalent to microsome by isopycnic centrifugation. These findings indicate that DNA polymerase-α in the germinal vesicle is released into the cytoplasm and binds to the endoplasmic reticulum when the germinal vesicle breaks down.