GTPase from rod outer segments: characterization by photoaffinity labeling and tryptic peptide mapping

The photoaffinity label [γ-³²P]8-N₃GTP has been used to identify GTP-binding components in highly purified preparations of GTPase from bovine rod outer segments. These preparations contain two major polypeptides of 37,000 and 39,000 daltons. In the presence of photolyzing radiation, [γ-³²P]8-N₃GTP is covalently attached to the 37,000 dalton polypeptide. Tryptic peptide mapping of this polypeptide indicates that it is highly related to the 39,000 dalton species that has been previously identified as a GTP-binding component.     

Separation of purine derivatives by polymer-bound nucleic acid bases

Separation of a variety of purine bases, which include 7-methyl derivatives, was studied by using polyethyleneimine-coated silicagel which bound hypoxanthine, cytosine or guanine moieties. The separation behavior seems to be related to the interaction of imidazole part of purine derivatives with the resins through hydrogen bonding.     

Identification of Chitinase and Osmotin-Like Protein as Actin-Binding Proteins in Suspension-Cultured Potato Cells

Cytoplasmic aggregation is an early resistance-associated eventthat is observed in potato tissues either after penetrationof an incompatible race of Phytophthora infestans, the potatolate blight fungus, or after treatment with hyphal wall components(HWC) prepared from P. infestans. In potato cells in suspensionculture, the number of cells with cytoplasmic aggregation increasedupon treatment with HWC, but such an increase was suppressedby treatment with cytochalasin D prior to treatment with HWC.This result suggested that cytoplasmic aggregation in culturedpotato cells might be connected with the association of actinfilaments. To identify the molecular basis of cytoplasmic aggregation,we purified actin and actin-related proteins by affinity chromatographyon a column of immobilized DNase I from cultured potato cellsand isolated proteins of 43 kDa, 32 kDa and 22 kDa. Analysisof the amino-terminal amino acid sequences indicated that the43 kDa, 32 kDa and 22 kDa proteins were potato actin, basicchitinase and osmotin-like protein, respectively. This conclusionwas supported by the results of Western blotting analysis ofthe 43 kDa and 32 kDa proteins with antibodies against actinand basic chitinase. Binding analysis with actin coupled toactin-specific antibodies and biotinylated actin suggested thatthe 32 kDa and 22 kDa proteins had actin-binding activity. Inaddition, examination of biomolecular interactions using anoptical biosensor confirmed the binding of chitinase to actin.These results imply the possibility that basic chitinase andosmotin-like protein might be involved in cytoplasmic aggregation,hereby participating in the potato cell's defense against attackby pathogen. (Received June 11, 1996; Accepted January 27, 1997)     

Suppression for the proliferation of fibroblasts by external DNA

Phase-contrast and fluorescence microscopic observation showed that DNA added in the cell-culture medium for fibroblasts localized just on the surface of fibroblasts. The DNA bound to fibroblasts was found to be eluted by treating with collagenase. The suppression for the proliferation of fibroblasts by external DNA was confirmed with microscopic observation for the cells cultured in the presence and absence of DNA. Proliferation of the cells decreased from 412 to 155% by the addition of DNA. These results indicate that DNA has an affinity for collagen, the most major extracellular-matrix produced by fibroblasts, and suppresses the growth of fibroblasts.     

New diterpenes from Porella perrottetiana

Perrottetianal A and perrottetianal B, two new diterpene dialdehydes and a new ent-labdane-type diterpene diol have been isolated from the liverwort Porella perrottetiana and their structures have been elucidated by the chemical and spectral evidence.     

Pinguisanin,pinguisanolide and β-pinguisenediol,three new pinguisane-type sesquiterpenes from Porella platyphylla

Three new pinguisane-type sesquiterpenes, pinguisanin, pinguisanolide and β-pinguisenediol, together with the previously known deoxopinguisone, have been isolated from a European liverwort, Porella platyphylla, and their structures have been established by the spectral evidence and some chemical transformations.     

Kinetics of photosynthetic membrane protein assembly in Rhodopseudomonas spheroides

Glycogen phosphorylase in cell-free extracts of Neurospora crassa is activated 10- to 15-fold by incubation with MgATP^2?. When the MgATP^2? is removed, the active form (a form) reverts to the inactive form (b form). The inactivation requires Mg²⁺ and is inhibited by NaF. The results confirm that Neurospora crassa glycogen phosphorylase exists in two interconvertible forms and strongly suggests that the interconversion is catalyzed by a kinase and phosphatase. The a form was partially purified. The enzyme has a molecular weight of 320,000. Uridine diphosphate glucose is a linear competitive inhibitor with respect to glucose-1-phosphate and a linear non-competitive inhibitor with respect to glycogen. Glucose-6-phosphate is a hyperbolic (partial) noncompetitive inhibitor with respect to all substrates in both directions. The b form of the enzyme in crude cell-free extracts is stimulated 2- to 3-fold by 5′-AMP. As the b form is purified, the 5′-AMP activation is diminished. The molecular weight of the partially purified “b” form was also 320,000.     

Significance of anaerobic preincubation of Helicobacter pylori for measuring metronidazole susceptibility by the Etest.

The Etest is widely used for measuring the susceptibility of Helicobacter pylori to metronidazole. By using 55 H. pylori isolates from 55 patients and a standard H. pylori strain, NCTC11637, we compared metronidazole susceptibility results obtained from the Etest with or without anaerobic preincubation to those obtained from the agar dilution method. Mueller Hinton agar plates supplemented with 5% horse blood were used for both methods. For the Etest, plates were incubated for 72 hr at 35 C under microaerophilic conditions after 0-, 4- or 24-hr periods of anaerobic preincubation. For the agar dilution method, the plates were incubated at the same microaerophilic conditions as those for the Etest. Without anaerobic preincubation for the Etest, 39 of the 56 (70%) H. pylori isolates were categorized as resistant to metronidazole (minimal inhibitory concentration>8 mg/liter), whereas only one of the 56 (1.8%) isolates was resistant according to the agar dilution method. The resistant and susceptible agreement rate was 32%. Four-hour anaerobic preincubation did not alter the readings of the Etest significantly. However, when the Etest was performed with 24-hr anaerobic preincubation, the number of isolates categorized as resistant was reduced to six (11%), improving the agreement rate to 91%. For measuring the metronidazole susceptibility of H. pylori by the Etest, 24-hr anaerobic preincubation is necessary to agree with the results obtained by the agar dilution test.