The genomic structure of two ancestral haplotypes carrying C4A duplications

Two major histocompatibility complex (MHC) ancestral haplotypes (AH) HLA A24, Bw52, C2C, BfS, C4A3 + 2, C4BQO, DRw15, DQw6 (52.1) and HLA A24, Cw7, B7, C2C, BfS, C4A3 + 3, C4B1, DR1, DQw5 (7.2), which occur with the haplotype frequencies of approximately 10% and 4% respectively in the Japanese population, carry duplicated C4A alleles by C4 allotyping. Southern blot analysis with Taq I indicated that the 52.1 AH has two C4 genes defined by 7.0 kilobase (kb) and 6.0 kb C4 hybridizing fragments but both encode C4A allotypes, being C4A3 and C4A2 respectively. The 7.2 AH carries two C4A3 and one C4B1 alleles and restriction lenght polymorphism (RFLP) analysis with Taq I showed that 6.0 kb and 7.0 kb fragments are in the proportion of 2:1. By pulsed field gel electrophoresis (PFGE) analysis, the lengths of the Pvul fragments carrying C4 and Cyp21 genes were approximately 390 kb for 52.1 and 440 kb to 7.2. The results indicate that the RFLP markers do not correlate with C4 isotype (A or B) or allotype and that the C4 gene copy number is a function of the number of genomic blocks containing C4 and Cyp21.     

Spectrophotometric characterization of phytochromes in dimorphic cocklebur seeds in relation to capacity for germination

Phytochrome was measured spectrophotometrically in different tissues of the upper (positively photoblastic) and lower (negatively photoblastic) seeds of the cocklebur (Xanthium pennsylvanicum Wallr.). Axial parts of the seeds, in particular parts of the radicle, contained high levels of phytochrome, while cotyledonary parts contained only low levels. These results were consistent with the distribution of the light-sensitive areas of the seeds that were associated with germination. Phytochrome levels in both types of dimorphic seeds increased gradually with increasing duration of dark imbibition for 4–8 h, then the rates of increase in levels of phytochrome accelerated. In both types of seed, some phytochrome was measurable even before imbibition. In the lower seeds, up to 20% of the phytochrome was occasionally observed as P_fr in samples imbibed in darkness for a short time (up to 12 h). A slight blue shift of the peak of P_T in the difference spectrum of phytochrome was observed in the case of lower seeds imbibed for 0–2 h. These results suggest that, to some extent, the lower axes contain dehydrated P_fr or intermediate(s) in the photoconversion of phytochrome. The dark reactions of P_fr were also examined in excised axes of both types of dimorphic seed after they had been pre-imbibed for 16 h in darkness. Dark destruction of P_fr was observed in both types of seed. In addition, net increases in levels of P_r were observed in the dark controls and in the samples irradiated with red light after the level of P_fr diminished. No ‘inverse’ dark reversion from P_r to P_fr was detected. Thus, after 16 h of imbibition, there were no differences in terms of properties of phytochrome between the two types of seed, and the different responses to light of upper and lower seeds might depend mainly on a difference in the physiological state of the two types of seed rather than the properties of phytochrome.     

Differences between the Surface Properties of the PR and PFR Forms of Native Pea Phytochrome, and Their Application to a Simplified Procedure for Purification of the Phytochrome

The ability of phytochrome from etiolated pea shoots (Pisumsativum L. cv. Alaska) to bind to various chromatographic adsorbentsand its mobility during non-denaturing electrophoresis wereexamined with phytochrome in either the red light-absorbingform (PR) or the far-red light-absorbing form (P_FR). Preferentialbinding of P_FR to modified hydrophilic polyvinyl resins, suchas butyl Toyopearl, phenyl Toyopearl, Blue Toyopearl (CibacronBlue F3G-A conjugated) and Red Toyopearl (Procion Red HE-3Bconjugated), was observed. A simplified method for purificationof native phytochrome was developed based on the propertiesof P_R and P_FR. P_FR bound preferentially to the hydrophobic adsorbents,to indicate that the surface of P_FR is more hydrophobic thanthat of P_R. A difference in net surface charges between P_R andP_FR was detected by an analysis based on the different mobilitiesof the two forms during non-denaturing polyacrylamide gel electrophoresisin gels prepared with various concentrations of polyacrylamide.The apparent molecular weights of P_R and P_FR, estimated fromthe analysis, were 378 and 419 kilodaltons, respectively. Thedifference suggests that a significant change in molecular shapeoccurs during the photoconversion. The differences in surfaceproperties of P_R and P_FR are discussed. (Received April 20, 1991; Accepted August 26, 1991)     

Involvement of C-terminal amino acids of a hyperthermophilic serine racemase in its thermostability

Extremophiles - Pyrobaculum islandicum is a hyperthermophilic archaeon that grows optimally at 95–100 °C. In the previous study, we extensively purified a serine racemase from...     

Simple and sensitive assay of zonisamide in human serum by high-performance liquid chromatography using a solid-phase extraction technique

A rapid and sensitive method for the assay of zonisamide in serum was developed using a solid-phase extraction technique followed by high-performance liquid chromatography. A 20-μl volume of human serum was first purified with a Bond-Elut cartridge column. Then, the methanol eluate was injected onto a reversed-phase HPLC column with a UV detector. The mobile phase was acetonitrile—methanol—distilled water (17:20:63, v/v) and the detection wavelength was 246 nm. The detection limit was 0.1 μg/ml in serum. The coefficients of variation were 4.2–5.6% and 5.1–9.1% for the within-day and between-day assays, respectively. This method can be used for clinical pharmacokinetic studies of zonisamide in serum even in infant patients with epilepsy.     

Alanine racemase from Helicobacter pylori NCTC 11637: purification, characterization and gene cloning

The Helicobacter pylori NCTC 11637 alanine racemase gene, alr1, was cloned based on a putative alanine racemase gene, alr, of H. pylori 26695. The protein, Alr1, was purified to homogeneity from Escherichia coli MB2795 cells harboring the alr1 gene. The protein exclusively catalyzes the conversion of l-alanine to the d-isomer with K(m) and V(max) values of 100 mM and 909 mumol min(-1) mg(-1), respectively. The values are 16-fold higher than those for the reaction in the reverse direction. The molecular weight of Alr1 is 42,000 by SDS-PAGE, and 68,000 by gel-filtration analysis. The optimal pH and temperature are pH 8.3 and 37 degrees C, respectively, in good accordance with the characteristics shown by the alanine racemase purified from H. pylori NCTC 11637 cells. Pyridoxal 5'-phosphate was suggested to be the cofactor. The physiological function of Alr1 is discussed regarding energy production in the microbial cells.     

A practical genome scan for population-specific strong selective sweeps that have reached fixation

Phenotypic divergences between modern human populations have developed as a result of genetic adaptation to local environments over the past 100,000 years. To identify genes involved in population-specific phenotypes, it is necessary to detect signatures of recent positive selection in the human genome. Although detection of elongated linkage disequilibrium (LD) has been a powerful tool in the field of evolutionary genetics, current LD-based approaches are not applicable to already fixed loci. Here, we report a method of scanning for population-specific strong selective sweeps that have reached fixation. In this method, genome-wide SNP data is used to analyze differences in the haplotype frequency, nucleotide diversity, and LD between populations, using the ratio of haplotype homozygosity between populations. To estimate the detection power of the statistics used in this study, we performed computer simulations and found that these tests are relatively robust against the density of typed SNPs and demographic parameters if the advantageous allele has reached fixation. Therefore, we could determine the threshold for maintaining high detection power, regardless of SNP density and demographic history. When this method was applied to the HapMap data, it was able to identify the candidates of population-specific strong selective sweeps more efficiently than the outlier approach that depends on the empirical distribution. This study, confirming strong positive selection on genes previously reported to be associated with specific phenotypes, also identifies other candidates that are likely to contribute to phenotypic differences between human populations.     

Phenotypic characterization of spontaneously mutated rats showing lethal dwarfism and epilepsy

We have characterized the phenotype of spontaneously mutated rats, found during experimental inbreeding in a closed colony of Wistar Imamichi rats. Mutant rats showed severe dwarfism, short lifespan (early postnatal lethality), and high incidence of epileptic seizures. Mutant rats showed growth retardation after 3 d of age, and at 21 d their weight was about 56% that of normal rats. Most mutant rats died without reaching maturity, and 95% of the mutant rats had an ataxic gait. About 34% of the dwarf rats experienced epileptic seizures, most of which started as 'wild running' convulsions, progressing to generalized tonic-clonic convulsions. At age 28 d, the relative weight of the testes was significantly lower, and the relative weight of the brain was significantly higher, in mutant than in normal rats. Histologically, increased apoptotic germ cells, lack of spermatocytes, and immature Leydig cells were found in the mutant testes, and extracellular vacuoles of various sizes were present in the hippocampus and amygdala of the mutant brain. Mutant rats had significantly increased concentrations of plasma urea nitrogen, creatinine, and inorganic phosphate, as well as decreased concentrations of plasma growth hormone. Hereditary analysis showed that the defects were inherited as a single recessive trait. We have named the hypothetically mutated gene as lde (lethal dwarfism with epilepsy).     

The RopGEF KARAPPO Is Essential for the Initiation of Vegetative Reproduction in Marchantia polymorpha

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Synthesis and evaluation of 4-(2-fluoro-4-[11C]methoxyphenyl)-5-((2-methylpyridin-4-yl)methoxy)picolinamide for PET imaging of the metabotropic glutamate receptor 2 in the rat brain

Metabotropic glutamate receptor 2 (mGluR2) has been suggested as a therapeutic target for treating schizophrenia-like symptoms arising from increased glutamate transmission in the human forebrain. However, no reliable positron emission tomography (PET) radiotracer allowing for in vivo visualization of mGluR2 in the human brain is currently available. In this study, we synthesized 4-(2-fluoro-4-[¹¹C]methoxyphenyl)-5-((2-methylpyridin-4-yl)methoxy)picolinamide ([¹¹C]1) and evaluated its potential as a PET tracer for imaging mGluR2 in the rodent brain. Compound 1, a negative allosteric modulator (NAM) of mGluR2, showed high in vitro binding affinity (IC₅₀: 26?nM) for mGluR2 overexpressed in human cells. [¹¹C]1 was synthesized by O-[¹¹C]methylation of the phenol precursor 2 with [¹¹C]methyl iodide. After the reaction, HPLC purification and formulation, [¹¹C]1 of 7.4?±?2.8?GBq (n?=?8) was obtained from [¹¹C]carbon dioxide of 22.5?±?4.8?GBq (n?=?8) with >99% radiochemical purity and 70?±?32?GBq/μmol (n?=?8) molar activity at the end of synthesis. In vitro autoradiography for rat brains showed that [¹¹C]1 binding was heterogeneously distributed in the cerebral cortex, striatum, hippocampus, and cerebellum. This pattern is consistent with the regional distribution pattern of mGluR2 in the rodent brain. The radioactivity was significantly reduced by self- or MNI-137 (a mGluR2 NAM) blocking. Small-animal PET studies indicated a low in vivo specific binding of [¹¹C]1 in the rat brain. The brain uptake was increased in a P-glycoprotein and breast cancer resistant protein double knockout mouse, when compared to a wild-type mouse. While [¹¹C]1 presented limited potential as an in vivo PET tracer for mGluR2, we suggested that it can be used as a lead compound for developing new radiotracers with improved in vivo brain properties.