Counterion displacement in the molecular evolution of the rhodopsin family

The counterion, a negatively charged amino acid residue that stabilizes a positive charge on the retinylidene chromophore, is essential for rhodopsin to receive visible light. The counterion in vertebrate rhodopsins, Glu113 in the third transmembrane helix, has an additional role as an intramolecular switch to activate G protein efficiently. Here we show on the basis of mutational analyses that Glu181 in the second extracellular loop acts as the counterion in invertebrate rhodopsins. Like invertebrate rhodopsins, UV-absorbing parapinopsin has a Glu181 counterion in its G protein-activating state. Its G protein activation efficiency is similar to that of the invertebrate rhodopsins, but significantly lower than that of bovine rhodopsin, with which it shares greater sequence identity. Thus an ancestral vertebrate rhodopsin probably acquired the Glu113 counterion, followed by structural optimization for efficient G protein activation during molecular evolution.     

Antioxidant activity of a Schiff base of pyridoxal and aminoguanidine

We recently reported that PL-AG, a Schiff base of pyridoxal and aminoguanidine, was more effective than aminoguanidine (AG), a well-known anti-diabetic-complication compound, in preventing nephropathy in diabetic mice and presented brief data indicating the antioxidant activity of the adduct. In the present study, we additionally investigated the inhibitory activity of PL-AG in comparison with that of AG against in vitro and in vivo oxidation. PL-AG was more potent than AG and reference compounds such as pyridoxal and pyridoxamine in any of the five antioxidant activities examined in vitro, i.e., hydrogen peroxide-scavenging, hydroxyl radical-scavenging, superoxide radical-scavenging, ascorbic acid-autoxidation inhibitory, and low-density lipoprotein (LDL)-oxidation inhibitory activities, the last two of which were assessed in the presence of Cu(2+). Unlike AG, PL-AG did not show the pro-oxidant activity. The inhibitory activity of PL-AG against lipid peroxidation in diabetic rats was higher than that of AG, for example, the amounts of malondialdehyde in erythrocytes (nmol/g hemoglobin; mean +/- SD) in normal, untreated diabetic, AG-treated diabetic, and PL-AG-treated diabetic rats were 3.53 +/- 0.35, 4.99 +/- 0.23, 4.65 +/- 0.45, and 4.06 +/- 0.35, respectively. A fluorescent substance different from PL-AG was found in the plasma and urine of rats treated with PL-AG. The chemical structure of this substance, i.e., oxidized PL-AG, was determined by a combination of nuclear magnetic resonance, mass, and infrared spectrometry. AG dramatically decreased the pyridoxal phosphate level in the diabetic rat liver, whereas PL-AG only moderately affected it. Our results indicate that the antioxidant activity of PL-AG is due to its chelation with transition metal ions and to scavenging of reactive oxygen species. They also suggest that PL-AG is more promising for the treatment of diabetic complications than AG.     

Regulated lens regeneration from isolated pigmented epithelial cells of newt iris in culture in response to FGF2/4

When a lens is removed from the newt eye, a new lens is regenerated from the pigmented epithelial cells of the dorsal iris, whereas the ventral iris never shows such an ability. It is important to clarify the nature of signaling molecules which act directly on the iris cells to accomplish lens regeneration from the iris and also to gain insight into the mechanism of dorso-ventral difference of the regeneration potential. To examine the effects of exogenous factors, we established an in vitro culture of reaggregates made from dissociated pigmented epithelial cells of dorsal or ventral halves of newt iris. Foci of depigmented cells appeared within the cell reaggregates, regardless of their origins, when the cell reaggregates were cultured with FGF2 or FGF4. In contrast, only the depigmented cells in the dorsal iris cell reaggregates underwent extensive proliferation and developed a lens with the synthesis of lens-specific crystallins, recapitulating the normal lens regeneration. On the other hand, neither FGF8, FGF10, EGF, VEGF, nor IGF promoted lens development from iris cell reaggregates. Consistent with the FGF-specific action, FGFR-specific inhibitor SU5402 suppressed the lens development from the cultured cell reaggregates. These results demonstrated that FGF2 or FGF4 is essential for the in vitro lens regeneration from the pigmented cells of the dorsal iris. In addition, these findings indicated that unequal competence in the dorsal and ventral iris to FGF2/4 contributes to the difference in lens forming ability between them.     

Hyperammonemia related to carnitine metabolism with particular emphasis on ornithine transcarbamylase deficiency

Carnitine status was evaluated in 8 patients with partial ornithine transcarbamylase (OTC) deficiency and 19 patients with secondary carnitine deficiency, who were used as positive references. Laboratory findings indicated that all patients with OTC deficiency had secondary carnitine deficiency especially in hyperammonemic attack. After L-carnitine administration in 2 patients with OTC deficiency, the number of attacks was significantly reduced in both cases.     

Surface Characteristics of Gram-Negative and Gram-Positive Bacteria in an Atomic Force Microscope Image

Bacterial images can be obtained rather easily with an atomic-force microscope (AFM) in the magnification range of 5,000 to 30,000 times without any pretreatment of the specimens for such observations as chemical fixation, dehydration or staining. The bacterial shapes or the presence of flagella can be clearly recognized in these magnification ranges. In addition, we were also able to distinguish between Gramnegative and Gram-positive bacteria based on the specific wavy surface appearance of the former. AFM could thus be a useful tool for the identification of bacteria in the resolution range between electron and light microscopy.     

Presence of human immunoglobulin G anti serum pancreatic elastase 1 autoantibodies and their influence on elastase 1 radioimmunoassay

Human immunoglobulin G (IgG) anti human pancreatic elastase 1 autoantibodies were detected in sera of patients with pancreatic disorders. The characteristics of these anti elastase 1 autoantibodies and their influence on radioimmunoassay (RIA) for elastase 1 were investigated. They were placed in the IgG class by the double antibody method, and most were assumed to be of a monoclonal type from their elution profiles in gel filtration analysis. The presence of autoantibodies in serum caused an increase in apparent elastase 1 values and a decrease in the recovery of elastase 1 exogenously added to the serum. These results suggest that elastase 1 immunoassay data for autoantibody positive sera can cause misjudgement of clinical stages of patients.