Photodesmosine,an isomer of desmosine obtained by photolysis of this amino acid in ultraviolet light

Desmosine and isodesmosine are two isomers representing the main cross-links of elastin. We describe a new isomer, photodesmosine, which is produced by the photolysis of desmosine at 254 nm. The mechanism of this photolysis is described and is shown to consist of two competing paths. After opening of the pyridium ring to give a tetrasubstituted aminoketone, this compound can either be hydrolysed to give lysine and a trisubstituted analogue of glutaconic aldehyde or undergo a recyclisation and rearomatisation to give a pyridium compound substituted in positions 1, 2, 3 and 4. An understanding of this mechanism is important in order to use photolysis as a specific method to break elastin cross-links. Although only desmosine and isodesmosine have been reported in purified elastin, the chromatographic properties of photodesmosine suggests that if other natural isomers exist in this protein they could be eluted from an ion-exchange resin at much earlier times than those observed in the case of the two already described cross-links.     

Stimulation by thyrotropin and cyclic AMP of the proliferation of quiescent canine thyroid cells cultured in a defined medium containing insulin

We have developed serum-free primary cultures of differentiated follicular dog thyroid cells which allow the study of the hormonal control of cell proliferation. The cooperation of insulin and increasing cellular cyclic AMP by thyrotropin triggers the DNA synthesis and the proliferation. Dog thyroid cells are an example of a system in which cyclic AMP is a sufficient signal to stimulate the proliferation in quiescent cells.     

Light-induced changes of fluorescence and absorbance in spinach chloroplasts at −40 °C

1. 1. Spinach chloroplasts were stored in the dark for at least 1 h, rapidly cooled to −40 °C, and illuminated with continuous light or short saturating flashes. In agreement with the measurements of Joliot and Joliot, chloroplasts that had been preilluminated with one or two flashes just before cooling showed a less efficient increase in the yield of chlorophyll a fluorescence upon illumination at −40 °C than dark-adapted chloroplasts. The effect disappeared below −150 °C, but reappeared again upon warming to −40 °C. Little effect was seen at room temperature in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), added after the preillumination.

2. 2. Light-induced absorbance difference spectra at −40 °C in the region 500–560 nm indicated the participation of two components, the socalled 518-nm change (P518) and C-550. After preillumination with two flashes the absorbance change at 518 nm was smaller, and almost no C-550 was observed. After four flashes, the bands of C-550 were clearly visible again.

3. 3. The fluorescence increase and the absorbance change at 518 nm showed the same type of flash pattern with a minimum after the second and a maximum at the fourth flash. In the presence of 100 μM hydroxylamine, the fluorescence response was low after the fourth and high again after the sixth flash, which confirmed the hypothesis that the flash effect was related to the so-called S-state of the electron transport pathway from water to Photosystem 2.

4. 4. The kinetics of the light-induced absorbance changes were the same at each wavelength, and, apart from the size of the deflection, they were independent of preillumination. Flash experiments indicated that the absorbance changes were a one-quantum reaction. This was also true for the fluorescence increase in dark-adapted chloroplasts, but with preilluminated chloroplasts several flashes were needed to approximately saturate the fluorescence yield.

5. 5. The results are discussed in terms of a mechanism involving two electron donors and two electron acceptors for System 2 of photosynthesis.

Hypochlorous acid generates N epsilon-(carboxymethyl)lysine from Amadori products

Since the accumulation of N^ε-(carboxymethyl)lysine (CML), a major antigenic advanced glycation end product, is implicated in tissue disorders in hyperglycemia and inflammation, the identification of the pathway of CML formation will provide important information regarding the development of potential therapeutic strategies for these complications. The present study was designed to measure the effect of hypochlorous acid (HOCl) on CML formation from Amadori products. The incubation of glycated human serum albumin (glycated-HSA), a model of Amadori products, with HOCl led to CML formation, and an increasing HOCl concentration and decreasing pH, which mimics the formation of these products in inflammatory lesions. CML formation was also observed when glycated-HSA was incubated with activated neutrophils, and was completely inhibited in the presence of an HOCl scavenger. These data demonstrated that HOCl-mediated CML formation from Amadori products plays a role in CML formation and tissue damage at sites of inflammation.     

Effect of Angeli's salt on the glutamate/glutamine cycle activity and on glutamate excitotoxicity in the hamster retina

Glutamate is the main excitatory neurotransmitter in the retina, but it is toxic when present in excessive amounts. It is well known that NO is involved in glutamate excitotoxicity, but information regarding the possibility that NO-related species could reciprocally affect glutamate synaptic levels was not previously provided. The dependence of glutamatergic neurons upon glia via the glutamate/glutamine cycle to provide the precursor for neurotransmitter glutamate is well established. The aim of the present work was to comparatively analyze the effect of nitroxyl and NO on the retinal glutamate/glutamine cycle in vitro activity. For this purpose, Angeli's salt (AS) and diethylamine NONOate (DEA/NO) were used as nitroxyl and NO donor, respectively. AS and DEA/NO significantly decreased retinal l-glutamate uptake and glutamine synthetase activity, but only AS decreased l-glutamine influx. Dithiothreitol prevented all the effects of AS and DEA/NO. The intravitreal injection of DEA/NO (but not AS) or a supraphysiological concentration of glutamate induced retinal histological alterations. Although AS could increase glutamate synaptic concentration in vitro, the histological alterations induced by glutamate were abrogated by AS. These results suggest that nitroxyl could regulate the hamster retinal glutamatergic pathway by acting through differential mechanisms at pre- and postsynaptic level.     

Photoactivation of the manganese catalyst of O2 evolution. I. Biochemical and kinetic aspects

Photosynthetic O₂ evolution requires a Mn complex which is activated by light. An analysis of this activation process yielded the following results:

1.

1. In any given illumination, the time course is first order, the rate being proportional to the number of inactive O₂-evolving System II trapping centers (the quantum yield being invariant).     

Biogenesis of chloroplast membranes X. Changes in the photosynthetic specific activity and the relationship between the light harvesting system and photosynthetic electron transfer chain during greening of Chlamydomonas reinhardi y-1 cells

Specific activities of photophosphorylation and light-dependent pH rise at different stages of the greening process, have been measured as a function of the illumination intensity.     

A distinct effect of transient and sustained upregulation of cellular factor XIII in the goldfish retina and optic nerve on optic nerve regeneration

Unlike in mammals, fish retinal ganglion cells (RGCs) have a capacity to repair their axons even after optic nerve transection. In our previous study, we isolated a tissue type transglutaminase (TG) from axotomized goldfish retina. The levels of retinal TG (TG(R)) mRNA increased in RGCs 1-6weeks after nerve injury to promote optic nerve regeneration both in vitro and in vivo. In the present study, we screened other types of TG using specific FITC-labeled substrate peptides to elucidate the implications for optic nerve regeneration. This screening showed that the activity of only cellular coagulation factor XIII (cFXIII) was increased in goldfish optic nerves just after nerve injury. We therefore cloned a full-length cDNA clone of FXIII A subunit (FXIII-A) and studied temporal changes of FXIII-A expression in goldfish optic nerve and retina during regeneration. FXIII-A mRNA was initially detected at the crush site of the optic nerve 1h after injury; it was further observed in the optic nerve and achieved sustained long-term expression (1-40days after nerve injury). The cells producing FXIII-A were astrocytes/microglial cells in the optic nerve. By contrast, the expression of FXIII-A mRNA and protein was upregulated in RGCs for a shorter time (3-10days after nerve injury). Overexpression of FXIII-A in RGCs achieved by lipofection induced significant neurite outgrowth from unprimed retina, but not from primed retina with pretreatment of nerve injury. Addition of extracts of optic nerves with injury induced significant neurite outgrowth from primed retina, but not from unprimed retina without pretreatment of nerve injury. The transient increase of cFXIII in RGCs promotes neurite sprouting from injured RGCs, whereas the sustained increase of cFXIII in optic nerves facilitates neurite elongation from regrowing axons.     

Chlorophyll fluorescence induction in anaerobic Scenedesmus obliquus

Fluorescence time curves (Kautsky effect) were studied in anaerobic Scenedesmus obliquus, with an apparatus capable of simultaneous recording of O₂ exchange, and far-red actinic illumination. Results, as interpreted in terms of electron transport reactions, suggest: In the course of becoming anaerobic, fluorescence induction undergoes a series of changes, indicating at least three different effects of the absence of O₂ on electron transport. (1) Immediately on removal of O₂, once the pool of intermediates between the two photo-systems is reduced by light reaction II, electron flow stops, resulting in high fluorescence yield and a cessation of O₂ evolution. O₂ appears to regulate linear electron flow and cyclic feedback of electrons to the intermediate pool. (2) An endogenous reductant formed anaerobically reduces the System II acceptors in the dark. The time course of this reduction is at least biphasic, indicative of inhomogeneity of the primary acceptor pool. Prolonged dark anaerobic treatment induces maximal initial fluorescence which decays rapidly in light and with a System I action spectrum. (3) Anaerobic treatment eventually results in deactivation of the oxidizing side of System II, limiting System II even when the acceptors are oxidized by System I pre-illumination.     

Functional homogeneity of P-700 in cyclic and non-cyclic electron transport reactions in thylakoids

The photoinduced turnover of P-700 (the reaction center chlorophyll a of photosystem I) in higher plant thylakoids was examined at room temperature by observation of the kinetics and amplitude of the transmission signal at 700 nm. The concentration of P-700 functional in cyclic and non-cyclic electron transfer reactions was compared. For the cyclic reactions mediated by N-methylphenazonium-p-methosulfate, 2,3,5,6-tetramethylphenylenediamine, 2,6-dichlorophenolindophenol and N,N,N′,N′-tetramethylphenylenediamine and non-cyclic reactions utilizing either methylviologen or NADP⁺ as acceptor, the illuminated steady-state concentration of P-700⁺ was shown to be similar. The data support the concept of a homogeneous pool of P-700 that is capable of interaction in both cyclic and non-cyclic electron transfer reactions and are consistent with previous data obtained in vivo.The amplitude and kinetics of the P-700 signal were found to be very dependent upon the composition of the reaction medium and differences were noted for turnover in the cyclic and non-cyclic reactions. Specifically, at white light saturation, the addition of low concentrations of divalent cations, such as Mg²⁺ or Ca²⁺, had no effect on the signal amplitude during the cyclic reactions, but, in confirmation of previous work, caused an attenuation of the signal amplitude during non-cyclic flow. At low light intensities, the divalent cations caused a similar reduction in redox level of P-700 in the steady-state during non-cyclic flow and also reduced the rate of P-700 photooxidation in the cyclic reactions. The concentration of divalent cation that reduced the signal amplitude of P-700⁺ during non-cyclic flow was compared with that required for the stimulation of the variable component of fluorescence, and it was shown to be similar with half maximal effects at 1 mM Mg²⁺. The observations confirm that divalent cations control non-cyclic electron transport by an activation of Photosystem II in addition to regulating the distribution of excitation energy between the two photosystems.     

Uncontrolled zinc- and copper-induced oligomerisation of the human complement regulator factor H and its possible implications for function and disease

Polymorphisms in factor H (FH), a major regulator of complement activation, and the accumulation of high zinc concentrations in the outer retina are both associated with age-related macular degeneration. FH is inhibited by zinc, which causes FH to aggregate. To investigate this, we quantitatively studied zinc-induced FH self-association by X-ray scattering and analytical ultracentrifugation to demonstrate uncontrolled FH oligomerisation in conditions corresponding to physiological levels of FH and pathological levels of zinc in the outer retina. By scattering, FH at 2.8-7.0 μM was unaffected until [Zn] increased to 20 μM, whereupon the radius of gyration, R_G, values increased from 9 to 15 nm at [Zn] = 200 μM. The maximum dimension of FH increased from 32 to 50 nm, indicating that compact oligomers had formed. By ultracentrifugation, size-distribution analyses showed that monomeric FH at 5.57 S was the major species at [Zn] up to 60 μM. At [Zn] above 60 μM, a series of large oligomers were formed, ranging up to 100 S in size. Oligomerisation was reversed by ethylenediaminetetraacetic acid. Structurally distinct large oligomers were observed for Cu, while Ni, Cd and Fe showed low amounts of oligomers and Mg and Ca showed no change. Fluid-phase assays showed reduced FH activities that correlated with increased oligomer formation. The results were attributed to different degrees of stabilisation of weak self-dimerisation sites in FH by transition metals. The relevance of metal-induced FH oligomer formation to complement regulation and age-related macular degeneration is discussed.     

Magnetic field effect on the chlorophyll fluorescence in Chlorella

The chlorophyll a fluorescence in Chlorella pyrenoidosa can be enhanced by 4–9% if the excitation light beam is parallel to an external magnetic field or decreased by 4–9% if the light beam is oriented perpendicular to a magnetic field of about 16 kgauss or more. These effects cannot be explained in terms of the small changes in light absorption which are also observed. It is suggested that these observations are due to a reorientation of pigment molecules in the magnetic field.     

Isolation and properties of particles containing the reactioncenter complex of Photosystem II from spinach chloroplasts

By density gradient centrifugation of the 80000 × g supernatant of digitonintreated spinach chloroplasts two main green bands and one minor green band were obtained. The purification and properties of the particles present in the main bands, which were shown to be derived from Photosystem I and Photosystem II, have been described previously; those of the particles in the minor fraction will be described in the present paper.

After purification, these particles show Photosystem II activity but are devoid of Photosystem I activity. They have a high chlorophyll a/chlorophyll b ratio and are enriched in β-carotene and cytochrome b₅₅₉. At liquid nitrogen temperature, photoreduction of C550 and photooxidation of cytochrome-b₅₅₉ can be observed. At room temperature, cytochrome b₅₅₉ undergoes slight photooxidation.

These properties indicate that this particle may be the reaction-center complex of Photosystem II. It is suggested that, in vivo, the Photosystem II unit is made up of a reaction-center complex and an accessory complex, the latter being found in one of the main green bands of the density gradient.     

Inhibitory effect of p-nitrothiophenol in the light on the Photosystem II activity of spinach chloroplasts

The treatment of spinach chloroplasts with p-nitrothiophenol in the light at acidic and neutral pH's caused specific inhibition of the Photosystem II activity, whereas the same treatment in the dark did not affect the activity at all. The photosystem I activity was not inhibited by p-nitrothiophenol both in the light and in the dark. The inhibition was accompanied by changes of fluorescence from chloroplasts. As observed at room temperature, the 685-nm band was lowered by the p-nitrothiophenol treatment in the light and, at liquid nitrogen temperature, the relative height of the 695-nm band to the 685-nm band increased and the 695-nm band shifted to longer wavelengths. The action spectra for these effects of p-nitrothiophenol on the activity and fluorescence showed a peak at 670 nm with a red drop at longer wavelengths. It was concluded that the light absorbed by Photosystem II is responsible for the chemical modification of chloroplasts with p-nitrothiophenol to causing the specific inhibition of Photosystem II.     

Flash-induced 515 nm absorbance change in chloroplasts with various granum contents

The 515 nm absorbance change was studied in mesophyll and bundle sheath chloroplasts of maize, which contain different amounts of grana. The amplitude of the 515 nm signal (induced by 3 μs flashes repeated at 4 s intervals) has shown a correlation with the granum content of the samples. However, upon addition of N-methylphenazonium methosulphate the 515 nm signal became independent of the amount of grana: in agranal thylakoids a large pool of silent Photosystem I was activated and, as a result, the amplitude of the 515 nm signal of agranal chloroplasts increased to the level exhibited by granal chloroplasts.These data show that the 515 nm absorbance change is not limited to small closed vesicles like grana, but in the presence of suitable electron donors single lamellae of bundle sheath chloroplasts can also be active.     

Excitation energy transfer and chlorophyll orientation in the green alga Chlamydomonas reinhardi

We have investigated the process of intermolecular excitation energy transfer and the relative orientation of the chlorophyll molecules in the unicellular green alga Chlamydomonas reinhardi. The principal experiments involved in vivo measurements of the fluorescence polarization as a function of the exciting-light wavelength in the presence and in the absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. We found that as the fluorescence lifetime increases upon the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea that the degree of fluorescence polarization decreases over the excitation region from 600 to 660 nm. This result, we argue, implies that a Förster mechanism of excitation energy transfer is involved for Photosystem II chlorophyll molecules absorbing primarily below 660 nm. We must add that our results do not exclude the possibility of a delocalized transfer process from being involved as well. Fluorescence polarization measurements using chloroplast fragments are also discussed in terms of a Förster transfer mechanism. As the excitation wavelength approaches 670 nm the fluorescence polarization is nearly constant upon the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.Experiments performed using either vertically or horizontally polarized exciting light show that the fluorescence polarization increases as the exciting light wavelength increases from 650 to 673 nm. This suggests the possibility that chlorophyll molecules absorbing at longer wavelengths have a higher degree of relative order. Furthermore, these studies imply that chlorophyll molecules exist in discrete groups that are characterized by different absorption maxima and by different degrees of the fluorescence polarization. In view of these results we discuss different models for the Photosystem II antenna system and energy transfer between different groups of optically distinguishable chlorophyll molecules.     

1-(3,4-Dihydroxy-5-Methoxyphenyl-3-Methylbut-2-Ene from The Liverworth Plagiochila Rutilans

Extraction of the liverwort Plagiochila rutilans afforded 1-(3,4-dihydroxy-5-methoxyphenyl)3-methylbut-2-ene, the structure of which was confirmed by synthesis.