Interaction between leukocytic elastase and Kunitz-type inhibitors from bovine spleen

The four Kunitz-type protease inhibitors purified from bovine spleen, which include the basic pancreatic trypsin inhibitor (BPTI), form stable complexes with human leukocytic elastase. The values of the affinity constants of these complexes are similar, in agreement with the great structural similarity of the four inhibitors, but are lower than those measured for the complexes with other serine proteases. Two main factors appear to be responsible for the stability of these complexes, i.e., hydrophobic interactions and ionization phenomena that take place during complex formation. These two factors have been analyzed in terms of the general model previously used for describing the interaction between the serine proteases and their natural inhibitors.     

Interaction among anion,cation and glucose transport proteins in the human red cell

Summary The time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to band 3 can be measured by the stopped-flow method. We have previously used the reaction time constant, _DBDS, to obtain the kinetic constants for binding and, thus, to report on the conformational state of the band 3 binding site. To validate the method, we have now shown that the ID₅₀ (0.3±0.1 m) for H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is virtually the same as the ID₅₀ (0.47±0.04 m) for H₂-DIDS inhibition of red cell Cl^– flux, thus relating _DBDS directly to band 3 anion exchange. The specific glucose transport inhibitor, cytochalasin B, causes significant changes in _DBDS, which can be reversed with intracellular, but not extracellular,d-glucose. ID₅₀ for cytochalasin B modulation of _DBDS is 0.1±0.2 m in good agreement withK _D =0.06±0.005 m for cytochalasin B binding to the glucose transport protein. These experiments suggest that the glucose transport protein is either adjacent to band 3, or linked to it through a mechanism, which can transmit conformational information. Ouabain (0.1 m), the specific inhibitor of red cell Na⁺,K⁺-ATPase, increases red cell Cl^– exchange flux in red cells by a factor of about two. This interaction indicates that the Na⁺,K⁺-ATPase, like the glucose transport protein, is either in contact with, or closely linked to, band 3. These results would be consistent with a transport proteincomplex, centered on band 3, and responsible for the entire transport process, not only the provision of metabolic energy, but also the actual carriage of the cations and anions themselves.     

Inhibitors of ribonucleotide reductase alter DNA repair in human fibroblasts through specific depletion of purine deoxynucleotide triphosphates

Hydroxyurea, deoxyadenosine, pyridine-2-carboxaldehyde thiosemicarbazone, pyrazoloimidazole, 3,5-diamino-1,2,4 triazole (guanazole), 3,4,5-trihydroxy benzohydroxamic acid and 3,4-dihydroxy benzohydroxamic acid were examined for their effects on cellular dNTP pools, DNA excision repair, DNA replication and deoxynucleoside uptake in human diploid fibroblasts. All 7 agents were effective inhibitors of the UV excision repair process in noncycling quiescent cells, but not in rapidly dividing log-phase cells. This differential effect clearly demonstrates dependency upon modulation of cellular purine dNTP pool levels at the level of the reductase. Repair synthesis is shown to be less sensitive to all 7 reductase inhibitors than is replicative synthesis. Studies on cellular uptake of labeled DNA precursors in inhibitor-treated cells support the notion that deoxynucleosides cannot channel into the replicative synthesis process whereas they are readily utilized at repairing sites.Abbreviations HU hydroxyurea - dA deoxyadenosine - TSC pyridine-2-carboxaldehyde thiosemicarbazone - IMPY pyrazoloimidazole - THBA 3,4,5-trihydroxy benzohydroxamic acid - DHBA 3,4-dihydroxy benzohydroxamic acid - UDS unscheduled DNA synthesis - dT thymidine - dNTP deoxynucleoside triphosphate     

Inhibition of Cytophaga U67 gliding motility by inhibitors of polypeptide synthesis

Gliding motility of Cytophaga U67 and several other cytophagas was inhibited by a growth-permissive concentration of chloramphenicol (50 g/ml). Several other inhibitors of polypeptide synthesis also demonstrated this effect. Short-term exposure to several of these inhibitors resulted in reversible inhibition of gliding by growing cells. In wet mounts chloramphenicol-grown cells demonstrated non-translocational tumbling. Electrophoretic patterns of polypeptides released by ethylenediaminetetraacetate treatment of control and chloramphenicol-grown cells were distinct. Gliding of a spontaneous mutant was resistant to chloramphenicol at 50 g/ml; its motility was inhibited at the growth-permissive concentration of 400 g/ml.     

Eigenschaften des Elektronentransportsystems von Mitochondrien des Protisten Acanthamoeba castellanii Neff. II. Reinigung und Eigenschaften der NADH-Akzeptor-Oxydoreduktase des Elektronentransportsystems

ZUSAMMENFASSUNG. Es wird die Reinigung einer mitochondrialen NADH-Akzeptor-Oxydoreduktase (EC. 1.6.99.3) über folgende Stufen beschrieben: fraktionierte Ammonsulfatfällung, Chromatographie an DEAE-Cellulose und Molekularsiebchromatographie an Sephadex G 100. Das gewonnene Enzympräparat erwies sich als elektrophoretisch rein. Die Michaeliskonstante für NADH als einziges Substrat liegt bei 6,9 × 10⁻⁵ M NADH. Ein ebenfalls isoliertes extramitochondriales Enzym underscheidet sich vom intramitochondrialen in drei voneinander unabhängigen Eigenschaften: Substratspezifität, elektrophoretiche Mobilität, Molekulargewicht. Das isolierte intramitochondriale Enzym verhält sich gegenüber Atmungsketteninhibitoren ähnlich wie isolierte Mitochondrien.
SYNOPSIS. Mitochondrial NADH reductase (EC. 1.6.99.3) was isolated from Acanthamoeba castellanii by the following steps: fractionation by (NH₄)₂SO₄ precipitation, DEAE-cellulose chromatography, and chromatography on Sephadex G 100 columns. Purity of the enzyme preparation was demonstrated by electrophoresis. With NADH as the sole substrate, the K _m= 6.9 × 10⁻⁵ M NADH. Extramitochondrial enzyme, also isolated from the ameba, differed from the mitochondrial one in substrate specificity, electrophoretic mobility, and molecular weight. The mitochondrial NADH reductase and isolated mitochondria were affected in the same way by respiratory chain inhibtors.     

C-Glycosylflavones from Zea mays that inhibit insect development

A new C-glycosylflavone isolated from corn silk inhibits the growth and development of the corn earworm, Heliothis zea. This new compound was shown to be a 2″-O-α-l-rhamnosyl-6-C-(6-deoxy-xylo-hexos-4-ulosyl)luteolin. Also found co-occurring in corn silk were minor amounts of the corresponding 6-C-glycosylated analogs of chrysoeriol and apigenin.     

Isolation of signaling mutants of tomato (Lycopersicon esculentum)

As a first step towards developing a genetic system for investigating signaling processes in plants, we have developed a screen for signaling mutants deficient in a wound response. We have isolated two mutants of tomato that lack detectable production of proteinase inhibitors induced systemically in leaves by wounding. The mutants are deficient in the induction of both proteinase Inhibitor I and proteinase Inhibitor II but can be induced to respond at near wild-type levels by methyl jasmonate, a known elicitor of inhibitor production in tomato. While completely deficient in systemic production of proteinase inhibitors, both mutants produce some proteinase inhibitor in wounded leaves. This evidence suggests the existence of two signaling pathways, one local and one systemic, that regulate the induction of proteinase inhibitor snythesis in response to wounding.     

FOXO3a反馈上调人表皮生长因子受体3表达介导HER2+乳腺癌细胞酪氨酸激酶抑制剂治疗耐受

近年来，酪氨酸激酶抑制剂（tyrosine kinase inhibitors，TKI）类药物治疗HER2+乳腺癌进展迅速，但出现治疗耐受仍是迫切需要解决的问题。本研究采用TKI（AEE788、Lapatinib）处理HER2+乳腺癌细胞BT474和SKBR3，发现HER3在mRNA和蛋白质水平上的表达均上调。MTS及克隆形成实验结果显示，siRNA干扰HER3的表达能够显著抑制BT474、SKBR3细胞的增殖，表明干扰HER3可增强细胞对TKI的敏感性。为进一步考察TKI促进HER3表达的可能机制，Western 印迹及免疫荧光检测发现，AEE788、Lapatinib能够上调FOXO3a的表达且促进其入核。干扰FOXO3a可逆转TKI对HER3的诱导作用，说明TKI通过激活FOXO3a上调HER3的表达。综上所述，FOXO3a反馈上调HER3表达介导HER2+乳腺癌细胞TKI治疗耐受。这一研究发现，为临床解决TKI治疗耐受提供一定的理论基础。     

Antiproliferative Effect of the Tripeptide PyroGlu-Phe-GlyNH2on Murine Melanocytes: Transitory Delay of Cell Growthin Vitroand the Cell Cycle Specificity

Cell growth and differentiation in melanocyte cell populations are regulated by a wide range of bioactive substances. Recently, the tripeptide pyroGlu-Phe-GlyNH₂which inhibits melanocyte growthin vitrowas identified in both murine nontransformed melanocytes and malignant melanoma cells. The present study was undertaken to investigate the cell cycle specificity as well as the growth inhibitory profile of the tripeptide after a single or repeated administration to melanocyte cultures. Dose-related effects of the peptide were studied using three different bioassay systems: estimation of cell number, DNA synthesis, and cell flux into mitosis. Growth of melanocyte cultures as well as melanocyte mitotic activity were found to be reduced significantly by the tripeptide at two separate dose levels (10⁻¹¹and 10⁻¹⁴–10⁻¹⁵M). Growth inhibition of melanocyte population did not last long: less than 36 h after the first and less than 24 h after the second peptide addition to the cultures. The level of DNA synthesis in melanocytes remained unchanged after a single peptide administration. The findings indicate that the tripeptide pyroGlu-Phe-GlyNH₂causes transitory delay of cell growth in cultured melanocyte population resulting from a reversible inhibition of melanocyte transition from the G₂-phase of the cell cycle into mitosis.     

Interaction of Zn2+ with the donor side of Photosystem II

The inhibitory effect of Zn²⁺ on photosynthetic electron transport was investigated in native and CaCl₂-treated (depleted in extrinsic polypeptides) Photosystem II (PS II) submembrane preparations. Inhibition of 2,6-dichlorophenolindophenol photoreduction by Zn²⁺ was much stronger in protein-depleted preparations in comparison to the native form. It was found that Ca²⁺ significantly reduced the inhibition in the native PS II preparations, as did Mn²⁺ in a combination with H₂O₂ in the protein-depleted counterparts. No other tested monovalent or divalent cations could replace Ca²⁺ or Mn²⁺ in the respective experiments. Diphenylcarbazide could partially relieve (40–45%) the inhibition in both types of preparations. The above indicates the presence of an active Zn²⁺ inhibitory site on the donor side of PS II. However, neither Ca²⁺ nor Mn²⁺ could completely prevent inhibition by high concentrations of Zn²⁺ (>1 mM). We propose that elevated levels of Zn²⁺ strongly perturb the conformation of the PS II core complex and might also affect the acceptor side of the photosystem.Abbreviations PMSF phenylmethanesulfonyl fluoride - MES 2-(N-morpholino)ethane sulphonic acid - Chl chlorophyll - PS II Photosystem II - DCIP 2,6-dichlorophenolindophenol - DPC sym-diphenylcabazide - DCBQ 2,5-dichlorobenzoquinone