Biochemical Study on the Critical Period for Treatment of the Mottled Brindled Mouse

Hemizygous mottled brindled mice (Mobr/y mice) were treated by subcutaneous injection of copper and were decapitated on postnatal day 14. Cytochrome c oxidase (COX) activity of the brain mitochondria in the mice given 10 micrograms of copper/g on day 4 or 7 showed significant increases compared with that of untreated Mobr/y animals, and these mice had no neurological symptoms. Mice given 10 micrograms of copper/g on day 12 showed neither increases in COX activity nor clinical improvement. The brain levels of copper, noradrenaline, and dopamine in the mice treated on day 12 were the same as those in animals treated on day 4 or 7. The in vitro activities of dopamine-beta-hydroxylase of the brain were also the same among the treated mice, irrespective of the date of treatment. The results indicate that delays in copper treatment produce irreversible changes in COX activity of the brain and lead to clinical unresponsiveness to treatment.     

The Effects of Ethylene on the Coordinated Synthesis of Multiple Proteins: Accumulation of an Acidic Chitinase and a Basic Glycoprotein Induced by Ethylene in Leaves of Azuki Bean, Vigna angularis

The ethylene-induced synthesis and accumulation of proteinswere studied in the primary leaves of azuki bean (Vigna angularis).Seven different proteins, designated AZ17, 23, 27, 32, 35, 36,42 according to their molecular masses, were synthesized andaccumulated in response to ethylene. AZ27 and AZ42 were purifiedto homogeneity and characterized. AZ27 was identified as anacidic chitinase and accumulated in the extracellular space.The sequence of the 40 N-terminal amino acids of AZ27 showedno similarity to that of a basic chitinase from bean and tobacco,but it was highly homologous to that of a chitinase from virus-infectedcucumber leaves. AZ42 was identified as a glycoprotein thataccumulated intracellularly. A search for proteins with sequenceshomologous to an internal sequence of 18 amino acids in AZ42was unsuccessful. Immunochemical examination revealed that auxinand abscisic acid enhanced the ethylene-induced accumulationof AZ27 but not of AZ42. In contrast, levels of AZ42 were notaffected by auxin or abscisic acid, but cytokinin suppressedthe accumulation of one of the doublet bands of AZ42. TranslatablemRNAs coding for AZ27 and AZ42 were not present in leaves thathad not been treated with ethylene, but levels of these mRNAsincreased after such treatment. (Received March 1, 1991; Accepted May 8, 1991)     

Precise localization of the human thyroxine-binding globulin gene to chromosome Xq22.2 by fluorescence in situ hybridization

The human thyroxine-binding globulin (TBG) gene has been localized to X chromosome (Xq22.2) by in situ hybridization using a biotinylated gDNA probe. This is consistent with previous mapping of the TBG gene to chromosome Xq21-q22.     

Presequence binding factor-dependent and -independent import of proteins into mitochondria.

A cytosolic protein factor(s) is involved in the import of precursor proteins into mitochondria. PBF (presequence binding factor) is a protein factor which binds to the precursor form (pOTC) of rat ornithine carbamoyltransferase (OTC) but not to the mature OTC, and is required for the mitochondrial import of pOTC. The precursors for aspartate aminotransferase and malate dehydrogenase as well as pOTC synthesized in a reticulocyte lysate were efficiently imported into the mitochondria. However, the precursors synthesized in the lysate depleted for PBF by treatment with pOTC-Sepharose were not imported. Readdition of the purified PBF to the depleted lysate fully restored the import. pOTC synthesized in the untreated lysate sedimented as a complex with a broad peak of around 9 S, whereas pOTC synthesized in the PBF-depleted lysate sedimented at an expected position of monomer (2.5 S). When the purified PBF was readded to the depleted lysate, pOTC sedimented as a complex of about 7 S. In contrast to most mitochondrial proteins, rat 3-oxoacyl-CoA thiolase is synthesized with no cleavable presequence and an NH2-terminal portion of the mature protein functions as a mitochondrial import signal. The thiolase synthesized in the PBF-depleted lysate could be efficiently imported into the mitochondria, and readdition of PBF had little effect on the import. The thiolase synthesized in the untreated, the PBF-depleted, or the PBF-readded lysate sedimented at an expected position of monomer (2.5 S). These observations provide support for the existence of PBF-dependent and -independent pathways of mitochondrial protein import.     

Genotoxicity of ptaquiloside, a bracken carcinogen, in the hepatocyte primary culture/DNA-repair test

The genotoxicity of ptaquiloside (PT), recently isolated from bracken fern and shown to be carcinogenic, was examined by means of the hepatocyte primary culture/DNA-repair test. PT elicited clear unscheduled DNA synthesis with a dose-response effect. The result indicates that PT is a genotoxic carcinogen.     

Amyloid β-Mediated Zn2+ Influx into Dentate Granule Cells Transiently Induces a Short-Term Cognitive Deficit

We examined an idea that short-term cognition is transiently affected by a state of confusion in Zn²⁺ transport system due to a local increase in amyloid-β (Aβ) concentration. A single injection of Aβ (25 pmol) into the dentate gyrus affected dentate gyrus long-term potentiation (LTP) 1 h after the injection, but not 4 h after the injection. Simultaneously, 1-h memory of object recognition was affected when the training was performed 1 h after the injection, but not 4 h after the injection. Aβ-mediated impairments of LTP and memory were rescued in the presence of zinc chelators, suggesting that Zn²⁺ is involved in Aβ action. When Aβ was injected into the dentate gyrus, intracellular Zn²⁺ levels were increased only in the injected area in the dentate gyrus, suggesting that Aβ induces the influx of Zn²⁺ into cells in the injected area. When Aβ was added to hippocampal slices, Aβ did not increase intracellular Zn²⁺ levels in the dentate granule cell layer in ACSF without Zn²⁺, but in ACSF containing Zn²⁺. The increase in intracellular Zn²⁺ levels was inhibited in the presence of CaEDTA, an extracellular zinc chelator, but not in the presence of CNQX, an AMPA receptor antagonist. The present study indicates that Aβ-mediated Zn²⁺ influx into dentate granule cells, which may occur without AMPA receptor activation, transiently induces a short-term cognitive deficit. Extracellular Zn²⁺ may play a key role for transiently Aβ-induced cognition deficits.     

Direct suppression of TCR-mediated activation of extracellular signal-regulated kinase by leukocyte protein tyrosine phosphatase, a tyrosine-specific phosphatase.

Leukocyte protein tyrosine phosphatase (LC-PTP)/hemopoietic PTP is a human cytoplasmic PTP that is predominantly expressed in the hemopoietic cells. Recently, it was reported that hemopoietic PTP inhibited TCR-mediated signal transduction. However, the precise mechanism of the inhibition was not identified. Here we report that extracellular signal-regulated kinase (ERK) is the direct target of LC-PTP. LC-PTP dephosphorylated ERK2 in vitro. Expression of wild-type LC-PTP in 293T cells suppressed the phosphorylation of ERK2 by a mutant MEK1, which was constitutively active regardless of upstream activation signals. No suppression of the phosphorylation was observed by LC-PTPCS, a catalytically inactive mutant. In Jurkat cells, LC-PTP suppressed the ERK and p38 mitogen-activated protein kinase cascades. LC-PTP and LC-PTPCS made complexes with ERK1, ERK2, and p38alpha, but not with the gain-of-function sevenmaker ERK2 mutant (D321N). A small deletion (aa 1-46) in the N-terminal portion of LC-PTP or Arg to Ala substitutions at aa 41 and 42 resulted in the loss of ERK binding activity. These LC-PTP mutants revealed little inhibition of the ERK cascade activated by TCR cross-linking. On the other hand, the wild-type LC-PTP did not suppress the phosphorylation of sevenmaker ERK2 mutant. Thus, the complex formation of LC-PTP with ERK is the essential mechanism for the suppression. Taken collectively, these results indicate that LC-PTP suppresses mitogen-activated protein kinase directly in vivo.     

Analysis of native and oxidized low-density lipoprotein oxysterols using gas chromatography—mass spectrometry with selective ion monitoring

SUMMARY

Cholesterol oxidation products have been demonstrated to possess a wide variety of biological properties and have been implicated in playing an important role in the development of atherosclerosis. We have developed an analytical method using capillary gas chromatography-mass spectrometry (GC-MS) for the analysis of cholesterol oxidation products in low-density lipoprotein (LDL). The method uses programmed multiple selected ion monitoring (SIM), providing enhanced sensitivity and accuracy of peak detection over full-scan mass spectra. The major oxidation products of cholesterol in oxidized LDL were identified as 7β-hydroxy-cholesterol and 7-keto-cholesterol. Minor products included 4β-hydroxy-cholesterol, 6β-hydroxy-cholesterol and cholesterol-5α,6α-epoxide. Native LDL contains 7-lathosterol, which is a biosynthetic precursor of cholesterol, as well as low levels of 7β-hydroxy-cholesterol and 7-keto-cholesterol. 7-Lathosterol was not detected in oxidized LDL. A time course oxidation of native LDL with 8 μM CuCl₂ demonstrated a rapid increase in 7β-hydroxy-cholesterol and 7-keto-cholesterol over the first 4 h. Cholesterol—5α,6α-epoxide, and β4-hydroxy- and 6β-hydroxy-cholesterol levels increased gradually, while 7-lathosterol decreased over the same period. This method was used to measure the levels of 7-lathosterol and cholesterol oxides in the LDL of 20 healthy subjects in order to establish the mean concentration and a reference range. This method can be used for the characterization and quantitation of oxysterols in native and oxidized LDL and may afford an additional index of oxidative modification of plasma lipoproteins.