High mobility group nonhistone chromosomal proteins also exist in Tetrahymena.

High mobility group (HMG) nonhistone chromosomal proteins have been shown to exist also in the ciliated protozoan Tetrahymena pyriformis. One or two histone-like components were extracted with 0.25 M HCl from the chromatin, in addition to five histone species. These proteins were also extracted selectively with 0.5 M HClO4, 0.35 M NaCl, or 4 mM spermidine, together with H1 histone, and were characterized as HMG proteins on the basis of the following criteria: high mobilities on polyacrylamide gel electrophoresis, relatively low molecular weights, amino acid compositions rich in lysine and glutamic acid, and relative contents in chromatin. This extends the distribution of the HMG proteins to all four eukaryotic kingdoms, and suggests the possibility that they have some universal role in chromatin structure and function.     

Cell expansion and microfibril deposition inClosterium ehrenbergii

Cells ofClosterium ehrenbergii expanded for about 4.5 hr after vegetative cell division and about 3 hr after sexual cell division. Short cells were produced by the sexual expansion. At the early stage of the vegetative and the sexual expansion, most microfibrils were deposited transversely to the cell axis on the inner surface of the new wall. Then, bundles of microfibrils, running in various directions, overlaid the transverse microfibrils already deposited, at the late stage of the both types of expansion. The pattern of microfibril deposition changed from the transverse to the bundled pattern about 4 hr after the vegetative and about 2 hr after the sexual cell division, respectively. The change of pattern of microfibril deposition seemed to be highly correlated with cessation of cell expansion.     

Kinetic study of isomerization of ferricytochrome c at alkaline pH

Kinetic studies of the isomerization reaction of horse heart ferricytochrome c between pH 8.5 and pH 12.1 have been carried out by using stopped-flow and rapid scanning stopped-flow techniques. Below pH 10, our results were in good agreement with the scheme proposed earlier (Davis, L. A., Schejter, A. and Hess, G. P. (1974) J. Biol. Chem. 249, 2624–2632). Above pH 10, another faster first-order process was observed, which suggested the existence of a transient species in the isomerization reaction between the species with and without a 695 nm band. The probable scheme of the isomerization reaction is considered to be

where H denotes a proton, the colored forms are the species predominant at neutral pH with a 695 nm band and the noncolored forms are the species without a 695 nm band. The transient species has a small 695 nm absorbance which suggests that the sixth ligand is still Met-80, although the protein conformation might be different from that at neutral pH.     

Studies on the sites in histones phosphorylated by adenosine 3':5'-monophosphate-dependent and guanosine 3':5'-monophosphate-dependent protein kinases.

Adenosine 3':5'-monophosphate-dependent protein kinase (protein kinase A) purified from silkworm pupae phosphorylated five major fractions of calf thymus histone, whereas guanosine 3':5'-monophosphate-dependent protein kinase (protein kinase G) purified from the same organism reacted preferentially with H1, H2A, and H2B histones. Amino acid analysis of the phosphopeptides which were obtained by proteolytic digestion revealed that both protein kinases A and G showed the abilities to phosphorylate the same serine hydroxyl groups in H1 and H2B histones. Both protein kinases reacted with Ser-38 in H1 histone. With H2B histone as substrate protein kinase A phosphorylated Ser-32 as well as Ser-36, whereas protein kinase G reacted preferentially with Ser-32 and the reaction with Ser-36 was very slow. H3 and H4 histones were practically inactive substrates for protein kinase G. Although H2A histone has not been analyzed, the evidence has raised a possibility that protein kinase G utilizes a portion of the substrate proteins for protein kinase A.     

Clinicopathological assessment of cancer/testis antigens NY-ESO-1 and MAGE-A4 in osteosarcoma

The cancer/testis antigens (CTAs), New York esophageal squamous cell carcinoma-1 (NY-ESO-1) and melanoma antigen gene (MAGE)-A4 are normally restricted to male germ cells but are aberrantly expressed in several cancers. Considering the limited information regarding their significance in osteosarcoma (OS), the purpose of this study was to determine the clinical significance of NY-ESO-1 and MAGE-A4 expression in OS. Nine patients with OS treated at Kindai University Hospital were included in the study. The median age was 27 years, and median follow-up period was 40 months. The specimens obtained at the time of biopsy were used to perform immunostaining for NY-ESO, MAGE-A4, p53, and Ki-67. The positive cell rates and positive case rates of NY-ESO, MAGE-A4, p53, and Ki-67 were calculated. The correlation between the positive cell rate of immunohistochemical markers was also calculated. The correlation between the positive cell rate of NY-ESO-1 or MAGE-A4 and tumor size or maximum standardized uptake (SUV-max) was also determined. The positive cell rates of NY-ESO-1 or MAGE-A4 in continuous disease-free (CDF) cases were also compared with those in alive with disease (AWD) or dead of disease (DOD) cases. The average positive cell rates of NY-ESO, MAGEA4, p53, and Ki-67 were 71.7%, 85.1%, 16.2%, and 14.7%, and their positive case rates were 33.3%, 100%, 44.4%, and 100%, respectively. The positivity rates of NY-ESO-1 and p53 were strongly correlated, whereas those of NY-ESO-1 and Ki-67 were moderately correlated. The MAGE-A4 and p53 positivity rates and the MAGE-A4 and Ki-67 positive cell rates were both strongly correlated. The NY-ESO-1 and MAGE-A4 positivity rates were moderately correlated. The positive correlation between the NY-ESO-1 positive cell rate and tumor size was medium, and that between the MAGE-A4 positivity rate and SUV-max was very strong. There was no significant difference in the positive cell rates of NY-ESO-1 or MAGE-A4 between CDF cases and AWD or DOD cases. Overall, our results suggest that NY-ESO-1 and MAGE-A4 may be involved in the aggressiveness of OS.Key words: New York esophageal squamous cell carcinoma-1 (NY-ESO-1), melanoma antigen gene (MAGE)- A4, osteosarcoma, prognosis, cancer/testis antigen (CTA), immunohistochemistry     

Quantitative effects of common genetic variations in the 3′UTR of the human LDL-receptor gene and their associations with plasma lipid levels in the Atherosclerosis Risk in Communities study

The low-density lipoprotein receptor (LDLR) plays a pivotal role in cholesterol homeostasis. However, the role of genetic variations in the 3′UTR of the LDLR in relation to plasma cholesterol has been largely understudied. Six SNPs, G44243A, G44332A, C44506G, G44695A, C44857T and A44964G, within the 5′ region of the 3′UTR fall into three common haplotypes, GGCGCA, AGCACG, and GGCGTA, occurring at frequencies of 0.45, 0.31 and 0.17, respectively, in Caucasians (n = 29) and 0.13, 0.13 and 0.38, respectively, in African Americans (n = 32), with three other haplotypes occurring at lesser frequencies. In a tissue culture based system, expression of a reporter gene carrying a 3′UTR that includes the 1 kb nucleotide sequences corresponding to the AGCACG or GGCGTA was 70 or 63%, respectively, of the same sequence with GGCGCA. Genotyping of two “haplotype tagging” SNPs, C44857T and A44964G, in the Atherosclerosis Risk in Communities (ARIC) study population showed that in Caucasians, but not in African Americans, the inferred TA haplotype had a significant LDL-cholesterol lowering effect. The adjusted LDL-cholesterol levels in the TA/TA diplotypes were lower by 6.10 mg/dl in men (P < 0.001) and by 4.63 mg/dl in women (P < 0.01) than in individuals with other diplotypes. Caucasian men homozygous for CA, in contrast, showed significantly higher LDL-cholesterol (P < 0.04), lower HDL-cholesterol (P < 0.02) and higher LDL/HDL ratios (P < 0.001). Thus our data shows that 3′UTR sequences that cause higher reporter gene expression in vitro are associated in Caucasians with plasma lipid profiles indicative of higher cardiovascular risk, suggesting that further studies of quantitative variants in the LDLR gene will be valuable. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

Developmental Changes in the NGF Content in the Brain of Young,Growing, Low-Birth-Weight Rats

The NGF content in each region of the brain of four-week-old rats was ranked in the decreasing order of cerebral cortex, hippocampus, cerebellum, midbrain/diencephalon, and pons/medulla ob-longata, and the NGF concentration, in the decreasing order of hippocampus, cerebral cortex, cerebellum, midbrain/diencephalon, and pons/medulla oblongata in both AFD and SFD groups. The NGF content and concentration in the cerebral cortex were about the same value at each age between those in the AFD and SFD groups. Those in the hippocampus were a little higher in the SFD group than in the AFD group at the ages of three and four weeks, unlike those in the other regions, where the values for the cerebellum, midbrain/diencephalon and pons/medulla oblongata tended to be somewhat higher in the AFD group than in the SFD group. The NGF concentrations in the hippocampus and cerebral cortex increased with growth: the concentration in the hippocampus at four weeks of age was about 4-fold of that at one week in the AFD group and about 5.7-fold of that at one week in the SFD group; and likewise the concentration in the cerebral cortex at four weeks of age was about 5.3-fold in the AFD group and about 7-fold in the SFD group. The NGF concentrations in the cerebellum decreased, and those in midbrain/diencephalon and pons/medulla oblongata hardly changed with growth in either AFD or SFD group. From these results NGF may have stronger implications for the neuronal growth in the hippocampus compared with those in the lower brain regions of the SFD rats.     

Antioxidative enzymes in seedlings of Nelumbo nucifera germinated under water

Dry seeds of anoxia-tolerant lotus ( Nelumbo nucifera Gaertn= Nelumbium speciosum Willd.) have green shoots with plastids containing chlorophyll, so photosynthesis starts even in seedlings germinated under water, namely hypoxia. Here we investigated antioxidative enzyme changes in N. nucifera seedlings responding to oxygen deficiency. The activity of superoxide dismutase (SOD; EC 1.15.1.1), dehydroascorbate reductase (DHAR; EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2) were lower in seedlings germinated under water (submerged condition) in darkness (SD seedlings) than those found in seedlings germinated in air and darkness (AD seedlings). In contrast, ascorbate peroxidase (APX; EC 1.11.1.11) activity was higher in SD seedlings and the activity of catalase (EC 1.11.1.6) and monodehydroascorbate reductase (MDAR; EC 1.6.5.4) in SD seedlings was nearly the same as in AD seedlings. When SD seedlings were exposed to air, the activity of SOD, DHAR and GR increased, while the activity of catalase and MDAR decreased. Seven electrophoretically distinct SOD isozymes were detectable in N. nucifera . The levels of plastidic Cu,Zn-SODs and Fe-SOD in SD seedlings were comparable with those found in AD seedlings, which may reflect the maintenance of green plastids in SD seedlings as well as in AD seedlings. These results were substantially different from those previously found in rice seedlings germinated under water.     

Linear polyubiquitin chains: A new modifier involved in NFκB activation and chronic inflammation including dermatitis

The ubiquitin conjugation system regulates a wide variety of biological phenomena, including protein degradation and signal transduction, by regulating protein function via polyubiquitin conjugation in most cases. Several types of polyubiquitin chains exist in cells, and the type of polyubiquitin chain conjugated to a protein seems to determine how that protein is regulated. We identified a novel linear polyubiquitin chain and the ubiquitin-protein ligase complex that assembles it, designated LUBAC. Both were shown to have crucial roles in the canonical NFκB activation pathway. This year, three groups, including our laboratory, identified SHARPIN as a new subunit of LUBAC. Of great interest, Sharpin was identified as a causative gene of chronic proliferative dermatitis in mice (cpdm), which is characterized by numerous inflammatory symptoms including chronic dermatitis, arthritis and immune disorders. Deletion of SHARPIN drastically reduces the amount of LUBAC and attenuates signal-induced NFκB activation. The pleomorphic symptoms of cpdm mice suggest that LUBAC-mediated NFκB activation may play critical roles in mammals and be involved in various disorders. A forward look into the linear polyubiquitin research is also discussed.Key words: ubiquitin, linear ubiquitination, NFκB, LUBAC, SHARPIN, cpdm, chronic dermatitis, TNFα     

Two Phenolic Amides in the Seed Balls of Sugar Beet (Beta vulgaris L. var. saccharifera Alefeld)

Ferredoxin-dependent sulfite reductase (Fd-SiR) (EC 1.8.7.1) was purified about 1136-fold, with a yield of 11%, from fresh thalli of Porphyra yezoensis by a procedure involving ammonium sulfate precipitation, DEAE-cellulose chromatography, Buty 1-Toyopearl chromatography, Sephadex G-100 gel filtration and ferredoxin-Sepharose affinity chromatography. The purified enzyme was apparently homogeneous, as judged on polyacrylamide disc gel electrophoresis, with a specific activity of 100 units/mg of protein. The molecular weight of the enzyme was estimated to be 70 kilodaltons by gel filtration. On subunit analysis by SDS-PAGE, a single band corresponding to molecular weight of 65 kilodaltons appeared. The purified enzyme (Fd-SiR) showed 5-times higher ferredoxin-dependent activity than methyl viologen-linked activity. In the oxidized form, the enzyme exhibited absorption maxima at 278, 390 (Soret band), 586 (a band) and 714 (CT band) nm, indicating that siroheme is involved in the catalysis of sulfite reduction. The absorbance ratios, A₃₉₀: A₂₁₈ and A₅₈₆ :A₃₉₀, were 0.32 and 0.31, respectively. A plot of the substrate (sulfite) and electron donor (ferredoxin) concentrations versus enzymatic (Fd-SiR) activity yielded sigmoidal curves, giving Hill coefficients («) of 2.3 (for sulfite) and 2.7 (for ferredoxin), respectively. Antibody against the isolated enzyme was raised in rabbits. Analysis of the antiserum by immunodiffusion suggested that it was specific against isolated Fd-SiR. Using the antiserum, dot immunoblotting was performed to determine the immunological similarity of Fd-SiRs from Porphyra yezoensis, Spirulina platensis, Brassica chinensis and Spinacia oleracea. The tests revealed that the four forms of assimilatory Fd-SiR have antigenic determinants in common.