Evidence for ion chain mechanism of the nonlinear charge transport of hydrophobic ions across lipid bilayers.

The conductivity across a lipid bilayer by tetraphenylborate anion is increased 10-fold on the photoformation of lipophilic porphyrin cations. The cations alone have negligible conductivity. This nonlinear photogenerated increase of ion conductivity is termed the photogating effect. Substitution of H by Cl in the para position of tetraphenylborate leads to a 100-fold enhancement of conductivity, whereas the dark conductivities for this and other substituted borates are the same. Moreover, the halo-substituted borates show a large enhancement of conductivity in the low concentration range (10(-8) M), whereas that of tetraphenylborate is small and space charge is negligible. The enhanced ion conductivity has great structural sensitivity to the structure of the anion, the cation, and the lipid, whereas the partition coefficient of all the borates and the concentration of photoformed cations are only slightly affected. The photogated ion transport has a twofold larger activation energy than transport in the dark. Time-resolved photocurrents and voltages demonstrate that the translocation rate of the porphyrin cation is also enhanced 100-fold by the Cl-borate anion but only 10-fold by the H-borate anion. For these reasons the nonlinear gating effect cannot be explained by electrostatics alone, but requires an ion chain or ion aggregate mechanism. Kinetic modeling of the photoinduced current with a mixed cation-anion ion chain can fit the data well. The photogating effect allows the direct study of ion interactions within the bilayer.     

Anchor-linked intermediates in peptide amide synthesis are caused by dimeric anchors on the solid supports

Cleavage and kinetic studies have been carried out using commercially obtained H-Tyr(tBu)-5-(4′-aminomethyl-3′,5′-dimethoxyphenoxy)valeric acid-TentaGelS (H-Tyr(tBu)-4-ADPV-TentaGelS) and H-Tyr (tBu)-4-ADPV-Ala-aminomethyl-resin (H-Tyr(tBu)-4-ADPV-AM-resin) prepared from commercially available resin and loaded with commercially available Fmoc-4-ADPV-OH amide anchor. Cleavage with pure trifluoroacetic acid (TFA) gave the intermediate H-Tyr-4-ADPV-NH₂, which was then degraded to H-Tyr-NH₂, and cleavage with TFA/dichloromethane (1:9) yielded H-Tyr-4-ADPV-NH₂ which could be isolated in preparative amounts. Cleavage reactions with ¹⁵N-labelled H-Ala-4-ADPV-[¹⁵N]-Gly-AM-resin yielded the intermediate H-Ala-4-ADPV-NH₂, which contained no ¹⁵N as demonstrated by ¹H-NMR. The analysis of the commercial Fmoc-4-ADPV-OH amide anchor showed the presence of Fmoc-4-ADPV-4-ADPV-OH as an impurity in high amounts. This dimeric anchor molecule is the cause of formation of the anchor-linked peptide intermediate obtained during the cleavage from the resin. The particularly high acid-lability of the amide bond between the two ADPV moieties was utilized to synthesize sidechain and C-terminally 4-ADPV protected pentagastrin on a double-anchor resin, and to cleave it using 5% trifluoroacetic acid in dichloromethane. This method may offer a new way for the synthesis of protected peptide amides with improved solubility to be used in fragment condensation.     

G2 phase cell cycle disturbance as a manifestation of genetic cell damage

The predominant cell cycle change induced by X-rays and clastogens in peripheral blood mononuclear cells is the accumulation of cells in the G2 phase of the cell cycle. We show that this accumulation consists of cells that are either delayed or arrested within the G2 phase. Since both X-rays and DNA crosslinking chemicals are known to damage DNA, the G2 phase inhibition caused by these agents is thought to be one of the primary manifestations of (unrepaired) DNA damage. This interpretation is supported by two additional findings. (1) Older individuals have elevated baseline levels of mononuclear blood cells that are delayed and/or arrested in the G2 phase of the cell cycle. This coincides with the increased chromosomal breakage rates reported for older individuals. (2) Irrespective of their age, individuals with inherited genetic instability syndromes (such as Fanconi anemia and Bloom syndrome) exhibit elevated G2 phase cell fractions. We show that the method used to detect such induced or spontaneous cell cycle changes, viz. BrdU-Hoechst flow cytometry, is a rapid and highly sensitive technique for the assessment of genetic cell damage.Dedicated to Professor Ulrich Wolf on the occasion of his 60th birthday     

大鼠胰腺γ-氨基丁酸免疫组织化学定位

本文用ABC—GDN免疫组织化学方法,研究了γ-氨基丁酸(Gamma—Aminobutyric Acid,GABA)在大鼠胰腺的定位和分布,并用相邻切片法,观察它与胰岛素的共存关系。结果发现GABA免疫反应阳性细胞主要分布于胰腺内分泌部(胰岛)。在外分泌部亦有少许分布。大部分胰岛细胞呈GABA免疫反应阳性,集中位于胰岛的中央部。相邻连续切片免疫染色证实GABA与胰岛素共存于胰岛B细胞中。外分泌部胰腺GABA免疫反应阳性细胞,呈零散分布于腺泡和导管上皮间。本文为进一步探讨GABA在胰腺的生理作用提供了形态学依据。     

Ce~（3＋）与G－肌动蛋白结合引起的构象变化及对聚合的影响

用Ａｅｄａｎｓ标记肌动蛋白单体Ｇ－Ａｃｔｉｎ上Ｃｙｓ３７４残基作为探针,研究了稀土离子Ｃｅ~（３＋）与Ｇ－Ａｃｔｉｎ的结合及引起的微构象变化。Ｃｅ~（３＋）在低浓度（Ｃｅ~（３＋）／Ａｃｔｉｎ摩尔比＜１）和Ｃａ~（２＋）竞争Ｇ－Ａｃｔｉｎ上二价离子的高亲合位点。Ｃｅ~（３＋）取代Ｃａ~（２＋）引起Ａｅｄａｎｓ荧光强度增强与Ｍｇ~（２＋）取代Ｃａ~（２＋）的结果相同。Ｃｅ~（３＋）／Ａｃｔｉｎ＞ｌ则导致Ａｅｄａｎｓ荧光强度下降。说明Ｃｅ~（３＋）在高低两种浓度条件下结合的位点及对Ｃｖｓ３７４的微构象的影响不同。时间分辩测得的Ａｅｄａｎｓ荧光寿命也支持这一结论。ＣＤ谱结果表明Ｃｅ~（３＋）／Ａｃｔｉｎ＜０．４,Ａｃｔｉｎ的二级结构增加,大于０．４又导致其失去。Ｃｅ~（３＋）－Ａｃｔｉｎ在有／无游离ＡＴＰ时用聚合液诱导的聚合结果表明,无游离ＡＴＰ时,极低浓度Ｃｅ~（３＋）促进聚合,高浓度虽有促进但有所减弱;有游离ＡＴＰ时,Ｃｅ~（３＋）／Ａｃｔｉｎ在实验范围内促进聚合。     

Functional Role of Amino-Terminal Serine16 and Serine27 of Gαz in Receptor and Effector Coupling

Abstract: The α subunit of G_z (α_z) harbors two N-terminal serine residues (at positions 16 and 27) that serve as protein kinase C-mediated phosphorylation sites. The cognate residues in the α subunit of G_t1 provide binding surfaces for the β₁ subunit. We used three serine-to-alanine mutants of α_z to investigate the functional importance of the two N-terminal serine residues. Wild-type or mutant α_z was transiently coexpressed with different receptors and adenylyl cyclase isozymes in human embryonic kidney 293 cells, and agonist-dependent regulation of cyclic AMP accumulation was examined in a setting where all endogenous α subunits of G_i were inactivated by pertussis toxin. Replacement of one or both serine residues by alanine did not alter the ability of α_z to interact with δ-opioid, dopamine D₂, or adenosine A₁ receptors. Its capacity to inhibit endogenous and type VI adenylyl cyclases was also unaffected. Functional release of βγ subunits from the mutant α_z subunits was not impaired because they transduced βγ-mediated stimulation of type II adenylyl cyclase. Constitutively active mutants of all four α_z subunits were constructed by the introduction of a Q205L mutation. The activated mutants showed differential abilities to inhibit human choriogonadotropin-mediated cyclic AMP accumulation in luteinizing hormone receptor-transfected cells. Loss of both serine residues, but not either one alone, impaired the receptor-independent inhibition of adenylyl cyclase by the GTPase-deficient mutant. Thus, replacement of the amino-terminal serine residues of α_z has no apparent effect on receptor-mediated responses, but these serine residues may be essential for ensuring transition of α_z into the active conformation.     

The use of percoll gradients, elutriator rotor elution, and mithramycin staining for the isolation and identification of intraerythrocytic stages of plasmodium berghei

Intraerythrocytic parasites of Plasmodium vinckei and Plasmodium berghei were separated according to their developmental stages using discontinuous Percoll gradients. Contaminating nucleated blood cells such as leukocytes were removed by elutriation centrifugation. The stages were unequivocally identified in smears using a newly developed DNA-specific staining procedure with mithramycin and fluorescence microscopy. This stain can also be used to detect parasites in human blood of very low parasitemias. The combination of methods described has many possible applications in immunologic and biochemical parasite research.     

Metabolism of prostaglandins in spontaneously hypertensive rats: NAD"-dependent 15-hydroxyprostaglandin dehydrogenase activity is decreased in kidney and increased in lung.

Renal, pulmonary and gastric NAD⁺-dependent 15-hydroxyprostaglandin dehydrogenase activities were determined in both spontaneously hypertensive and normotensive rats at 6 and 12 weeks of age. Renal enzyme activity in hypertensive rats was only 30–40% of that present in normotensive controls at both ages. In contract, pulmonary enzyme activity in hypertensive animals was twice as active as that in normal controls. There was no significant difference in gastric enzyme activity. NAD⁺-dependent 9-hydroxyprostaglandin dehydrogenase activity, the enzyme responsible for the conversion of vasoinactive PGF metabolites to PGE metabolites, also failed to show any difference in two types of rat kidneys. The results indicate that, in hypertension, prostaglandin inactivation is impaired in kidney but is facilitated in lung.     

Effect of alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, on polyamine levels in rat tissues.

α-Difluoromethylornithine (RMI 71.782), an enzyme-activated irreversible inhibitor of ornithine decarboxylase (E.C. 4.1.1.17) $\text{in vitro}$, causes a rapid, long-lasting, dose-dependent decrease of ornithine decarboxylase activity in prostate and, to a lesser extent, in thymus and testis of rats when injected intraperitoneally. Repeated doses (100 mg/kg or 1 g/kg) of α-difluoromethylornithine given to rats for two weeks markedly decreased polyamine concentrations in several rat tissues and selectively slowed down growth of ventral prostate and of thymus.     

Site-specific glycation of lens crystallins by ascorbic acid.

The oxidation of ascorbic acid leads to the formation of several compounds which are capable of reacting with protein amino groups via a Maillard reaction. Radioactivity from [1-14C]ascorbic acid was linearly incorporated into lens crystallins over a 10 day period in the presence of NaCNBH3. This rate of incorporation was 6-7-fold more rapid than that obtained with [14C]glucose under the same conditions. SDS-PAGE showed a linear incorporation into all the crystallin subunits. [1-14C]Ascorbic acid-label led alpha-crystallin was separated into its component A and B subunits, and each was digested with chymotrypsin. HPLC peptide analysis showed a differential labelling of the various lysine residues. Analysis of the peptides by mass spectrometry allowed the identification of the sites and the extent of modification. These values ranged from 6% for Lys-78 to 36% for Lys-11 in the A subunit and from 5% for Lys-82 to an average of 38% for the peptide containing Lys-166, Lys-174 and Lys-175 in the B subunit. Amino acid analysis demonstrated a single modification reaction producing N epsilon-(carboxymethyl)lysine. This agreed with the mass increase of 58 observed for each modified peptide.