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The primary photochemical event in rhodopsin is an 11-cis to 11-trans photoisomerization of its retinylidene chromophore to form the primary intermediate photorhodopsin. Earlier picosecond studies have shown that no intermediate is formed when the retinal 11-ene is fixed through a bridging five-membered ring, whereas a photorhodopsin-like intermediate is formed when it is fixed through a flexible seven-membered ring. Results from a rhodopsin analog formed from a retinal with locked 11-ene structure through the more flexible eight-membered ring (Ret8) are described. Incubation of bovine opsin with Ret8 formed two pigments absorbing at 425 nm (P425) and 500 nm (P500). P425, however, is an artifact because it formed from thermally denatured opsin or other proteins and Ret8. Excitation of P500 with a picosecond green pulse led to formation of two intermediates corresponding to photo- and bathorhodopsins. These results demonstrate that an appearance of early intermediates is dependent on the flexibility of the 11-ene and that the photoisomerization of P500 proceeds by stepwise changes of chromophore-protein interaction, which in turn leads to a relaxation of the highly twisted all-trans-retinylidene chromophore in photorhodopsin.

Duocarmycins have been reported to derive their potent antitumor activity through a sequence-selective minor groove alkylation of N3 adenine in double-stranded DNA. We have used gel mobility shift assays to detect proteins that bind to DNA treated in vitro with duocarmycin SA and identified a protein, named duocarmycin-DNA adduct recognizing protein (DARP), which binds with increased affinity to duocarmycin-damaged DNA. Examination with partially purified DARP revealed that the protein recognized not only the DNA adduct of structurally related drug, CC-1065, but unexpectedly, the protein also recognized the DNA adduct of another chemotype of minor groove binder, anthramycin. These results demonstrate that DARP recognizes the structural alteration of DNA induced by these potent DNA-alkylating drugs, suggesting the possibility that the protein might modulate the antitumor activity of these drugs.

We have developed an enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies against Helicobacter pylori (HP) using purified HP urease as an antigen. The urease was purified from ultrasonicated extract of HP by NaCl linear gradient system on DEAE-Sepharose 4B chromatography. Two molecular weight bands, 65kD and 27kD were observed on a SDS-PAGE gel in the purified urease sample. The urease antigen did not crossreact to rabbit antibodies prepared against Campylobacter coli and Campylobacter jejuni. Out of 93 gastric biopsy patients, sixty nine patients (74.2%) were positive in HP culture test. Serum HP antibody titers (AU: arbitrary unit) of HP culture positive and negative patients were 42.9 +/- 47.4 and 16.7 +/- 25.7 (mean +/- SD), respectively (p0.05). The ELISA system have sensitivity of 72.5% and specificity of 70.8%. We believe that the ELISA system is useful for diagnosis and monitoring of HP infection.

Erythropoietin (Epo) exerts its effects by binding specific receptors on the surface of reactive cells. However, the signal transduction system after binding has not been well described. To develop a system to analyze the steps of signal transduction, we transfected the human c-fos-enhancer/promoter linked with the Photinus pyralis luciferase gene (pfosluc2) into a murine erythroleukemia cell line ELM-I-1, in which we previously showed that c-fos mRNA is rapidly induced upon Epo-stimulation. A stable transfectant was obtained. The cells transfected with pfosluc2 were stimulated with Epo and luciferase activity in the cells was measured as light intensity. The light intensity integrated for 2 min (LI2.0) was 3202 +/- 80 unit/1.5 x 10(5) cells before stimulation. This increased up to 5869 +/- 321 unit/1.5 x 10(5) cells by incubating the cells with 5 U/ml Epo for 2 h. After Epo stimulation, light intensity began to increase at 30 min, reached a peak (about 1.8 times the basal level) at 120 min, and then gradually dropped. The effect of Epo was dose-dependent; significant action occurred at as low as 0.5 U/ml, with a maximum at 5 U/ml. A similar response was observed when the cells were stimulated with interleukin-3 (IL-3) although the response was apparently lower than that with Epo. It was also found that IL-3 had an additive action with Epo on c-fos activity in this system. Thus, the above method was proven to be simple, rapid and sensitive enough to use to determine the early phase of signal transduction of Epo.

Pancreatic islet cells of rats obtained by the collagenase digestion method were preserved from 7 to 10 days at -80 degrees C in a deep freezer or at -196 degrees C in liquid nitrogen with Dulbecco modified Eagle's medium, supplemented with 10% or 20% DMSO (dimethyl sulfoxide); thawing was performed in a 37 degree C water bath. Cryopreserved islets were morphologically almost intact, and possessed approximately 50% of the insulin secretion activity of the control groups. About 300 pancreatic islets preserved at -196 degrees C with 20% DMSO were transplanted into the portal veins of streptozotocin-induced diabetic rats. Rats recovered from the diabetic state, and the normalized condition was maintained up to 20 weeks, although 4 weeks were needed before blood and urine glucose reached normal levels after transplantation. Intact B cells were found in the transplanted islet cell masses in the liver of the recipients, but B cells of the recipient's pancreases (streptozotocin-treated rats) showed a marked decrease, as well as degenerative changes.