Comparison of the electron spin polarized spectrum found in plant photosystem I and in iron-depleted bacterial reaction centers with time-resolved K-band EPR; evidence that the photosystem I acceptor A1 is a quinone

The suggestion that the electron acceptor A₁ in plant photosystem I (PSI) is a quinone molecule is tested by comparisons with the bacterial photosystem. The electron spin polarized (ESP) EPR signal due to the oxidized donor and reduced quinone acceptor (P ₈₇₀ ⁺ Q^-) in iron-depleted bacterial reaction centers has similar spectral characteristics as the ESP EPR signal in PSI which is believed to be due to P ₇₀₀ ⁺ A ₁ ^- , the oxidized PSI donor and reduced A₁. This is also true for better resolved spectra obtained at K-band (24 GHz). These same spectral characteristics can be simulated using a powder spectrum based on the known g-anisotropy of reduced quinones and with the same parameter set for Q^- and A₁ ^-. The best resolution of the ESP EPR signal has been obtained for deuterated PSI particles at K-band. Simulation of the A₁ ^- contribution based on g-anisotropy yields the same parameters as for bacterial Q^- (except for an overall shift in the anisotropic g-factors, which have previously been determined for Q^-). These results provide evidence that A₁ is a quinone molecule. The electron spin polarized signal of P₇₀₀ ⁺ is part of the better resolved spectrum from the deuterated PSI particles. The nature of the P₇₀₀ ⁺ ESP is not clear; however, it appears that it does not exhibit the polarization pattern required by mechanisms which have been used so far to explain the ESP in PSI.Abbreviations hf hyperfine - A₀ A₀ acceptor of photosystem I - A₁ A₁ acceptor of photosystem I - Brij-58 polyoxyethylene 20 cetyl ether - CP1 photosystem I particles which lack ferridoxin acceptors - ESP electron spin polarized - EPR electron paramagnetic resonance - I intermediary electron acceptor, bacteriopheophytin - LDAO lauryldimethylamine - N-oxide, P₇₀₀ primary electron donor of photosystem I - PSI photosystem I - P₇₀₀ ^T triplet state of primary donor of photosystem I - P₈₇₀ primary donor in R. sphaeroides reaction center - Q quinore-acceptor in photosynthetic bacteria - RC reaction center     

A critical evaluation of the use of filipin-permeabilized rat hepatocytes to study functions of the endoplasmic reticulum in situ

We have used transmission (TEM) and scanning electron microscopy (SEM) and leakage of lactate dehydrogenase (LDH; EC 1.1.1.27) to evaluate two published procedures which use filipin to render isolated rat hepatocytes permeable to ionic substrates. Cells treated by the procedure of Jorgenson and Nordlie retained less than 10 per cent of their LDH. TEM revealed severe damage to the internal structure of these cells, which included swelling, disintegration and extensive vesicularization of the endoplasmic reticulum (ER). Hepatocytes treated with filipin by the procedure of Gankema et al. retained 65-75 per cent of their LDH and displayed incomplete but highly variable permeability to Trypan blue. SEM revealed the loss of microvilli, other signs of swelling, and the presence of large lesions in the plasma membrane. TEM revealed signs of cell swelling, but the nuclei and the mitochondria were only moderately altered. The rough ER was not swollen, but significant fragmentation was evident and characteristic stacks of lamellar ER were never seen. We conclude that useful information about the functions of the ER in situ cannot be obtained from studies of filipin-treated cells. Our results indicate that retention of LDH is not a sufficient criterion of preservation of cell morphology and that staining with Trypan blue may significantly underestimate the permeability of cells to small ionic metabolites.     

Meloidogyne californiensis n. sp. (Nemata: Meloidogyninae), Parasitic On Bulrush,Scirpus robustus Pursh

Meloidogyne californiensis n. sp. is described and illustrated from bulrush Scirpus robustus in California. LM and SEM studies revealed that this species differs from other known species in the genus Meloidogyne especially by the prominent posterior cuticular protuberances in the female, the distinct shape of the perineal pattern which is marked by one prominent stria in the perineum, indistinct lateral lines, many broken discontinuous striae on both sides of the arch, and the excretory pore being located posterior to stylet base. Second-stage juveniles 448-628 μm long, stylet length 11-13 μm, styler delicate, with small knobs sloping posteriorly, cephalic region with 2 or 3 annuli, and inflated rectum. Males vary greatly in size (712-1,952 μm), stylet length 18-28 μm (mean 22 μm), cephalic region slightly set off the body with two or three annuli, spear heavy with massive rounded knobs, lateral field marked by four areolated incisures as seen by SEM.     

Tubular invaginations in cerebral endothelium and their relation to smooth-surfaced cisternae in hagfish (Myxine glutinosa)

Summary The organization of vesicular profiles in the endothelium of cerebral capillaries of the hagfish, Myxine glutinosa, has been reinvestigated. Judged from random thin sections the endothelial cells contain numerous vesicles and tubules, in contrast to brain endothelia of most other vertebrates. However, three-dimensional reconstructions based on ultrathin serial sections (thickness 18 nm) showed that the profiles represent a system of irregular tubular invaginations of the cell membrane, comparable to the vesicular invaginations demonstrated in extracerebral capillary endothelia of frogs and rats. In addition, smooth-surfaced cisternae were present in close relation to the invaginations. The function of endothelial invaginations is unknown. They do not transport macromolecules, because the blood-brain barrier is practically impermeable to proteins. However, since the system of the invaginations and smooth-surfaced cisternae is structurally similar to the system of caveolae and sarcoplasmic reticulum in smooth muscle cells, a common function seems likely. It is proposed that endothelial invaginations and smooth-surfaced cisternae are involved in regulation of cytosolic Ca⁺⁺-concentration.     

Arterial supply of the choriocapillaris of anuran amphibians (Rana temporaria,Rana esculenta)

Summary The pattern of the vascular supply to the choroid of the frog eye was studied in toto with the use of the injection-replication-SEM technique. The choroid of anuran amphibians is composed mainly of the choriocapillaris. In both species studied (Rana temporaria, Rana esculenta), an independent arterial supply to the choriocapillaris supplemented that from the ciliary arteries. This additional vascular route arises from the optic artery, a separate branch of the arteria infundibularis superficialis. The optic artery, accompanied by its vein within the vascular sheath of the optic nerve, joins the rich arterial capillary network of the choriocapillaris and supplies the posterior pole of the ocular bulb. The superficial capillary network displays a dense collar around the entrance of the optic nerve into the eye and is composed of a circular meshwork of small capillaries, several layers deep. More peripherally, however, it becomes single layered. This capillary network, as a whole, establishes numerous connections with the adjacent choriocapillaris at the posterior pole of the ocular bulb. In anuran amphibians the complex arrangement of both arterial systems supporting the choriocapillaris may be regarded as a more complete equivalent of the short posterior ciliary arteries of mammals.     

The superficial vascular hyaloid system in the eye of the frogs,Rana temporaria and Rana esculenta

Summary The angioarchitecture of the superficial vascular hyaloid system (membrana vasculosa retinae) of the frog eye was studied by means of scanning electron microscopy of vascular corrosion casts. The terminal vessels form a single-layered sheath intimately adjacent to the vitreal side of the avascular retina. The hyaloid system is subdivided by the ventral venous trunk into three central areas: the dorsal, the temporo-ventral, and the naso-ventral area. Toward the ora serrata, the hyaloid system is bordered by an arterial ring, and by nasal and temporal venous branches forming more or less complete hemicircles. A vascular zone composed of several tongue-like sectors establishes an inter-connection between the peripheral vascular rings and the central areas of the fundus. The arterial blood is supplied from the arterial ring. The drainage of the hyaloid system is provided via two routes: (1) the Y-shaped ventral trunk collects blood from the central areas, (2) the two peripheral venous branches drain the tongue-like sectors. The vessels within the dorsal area follow preferentially a dorso-ventral meridional direction. This densely capillarized territory corresponds in localization to the area centralis retinae. The ultrastructure of microvessels of the hyaloid system is characterized by features typical for capillaries of the central nervous system.     

Immunocytochemical localization of lactoferrin in human neutrophils

Summary The subcellular localization of lactoferrin in human neutrophils was studied by an electron-microscopic immunoperoxidase method. This molecule was detected in small granules of blood polymorphonuclear leukocytes. A morphometrical analysis showed that there was no significant difference in the mean size between lactoferrin-positive and myeloperoxidase-negative granules. In contrast, the mean size of myeloperoxidase-positive granules was significantly larger than that of lactoferrin-positive granules. This indicates that lactoferrin is contained in the myeloperoxidase-negative, secondary, granules of human neutrophils. In immature bone marrow mononuclear neutrophils, lactoferrin was present in cytoplasmic granules of somewhat larger size than lactoferrin-positive granules of polymorphonuclear leucocytes. A morphometrical study showed that the mean size of lactoferrin-positive granules was significantly greater in immature bone marrow cells than in polymorphonuclear leucocytes. This indicates that lactoferrin-positive granules decrease in size as the cells mature. Besides cytoplasmic granules, lactoferrin was demonstrated in the Golgi complex and a part of the rough endoplasmic reticulum of immature bone marrow neutrophils, probably myelocytes and early metamyelocytes. These results show that lactoferrin is synthesized and packed into secondary granules in immature bone marrow neutrophils and therefore that the secondary granules are a type of secretory granule.     

Low temperature delays development of photosystem II activity in winter rye leaves

The ability of leaves to acclimate photosynthetically to low temperature was examined during leaf development in winter rye plants ( Secale cereale L. cv. Puma) grown at 20°C or at 6°C. All leaves grown at 6°C exhibit increased chlorophyll (Chl) levels per leaf area, higher rates of uncoupled, light-saturated photosystem I (PSI) electron transport, and slower increases in photosystem II (PSII) electron transport capacity, when compared with 20°C leaves. The stoiehiometry of PSI and PSII was estimated for each leaf age class by quantifying Chl in elcctrophorctic separations of Chl-protein complexes. The ratio of PSII/PSI electron transport in 20°C leaves is highly correlated with the ratio of core Chl a -proteins associated with PSII (CPa) to those associated with PSI (CP1). In contrast, PSII/PSI electron transport in 6°C leaves is not as well correlated with CPa/CP1 and is related, in part, to the amount and organization of light-harvesting Chl a/b -proteins associated with PSII. CPa/CP1 increases slowly in 6°C leaves, although the ratio of CPa/CP1 in mature 20°C and 6°C leaves is not different. The results suggest that increased PSI activity at low temperature is not related to an increase in the relative proportion of PSI and may reflect, instead, a regulatory change. Photosynthetic acclimation to low environmental temperature involves increased PSI activity in mature leaves shifted to 6°C. In leaves grown entirely at 6°C, however, acclimation includes both increased PSI activity and modifications in the rate of accumlation of PSII and in the organization of LHCII.     

Effect of abscisic acid on the photosynthetic oxygen evolution in barley chloroplasts

In vivo effect of abscisic acid (ABA) on photosynthetic oxygen evolution was investigated in barley chloroplasts. The most important kinetic parameters of O₂-producing reactions were changed. The results show inhibition of the O₂-flash yields at ABA concentrations of 10 mol/l and 100 mol/l and an increase in the degree of damping of the oscillations. ABA has a marked effect on the distribution of the oxygenevolving centers in S₀ and S₁ states and on sum of the centers (S₀+S₁) estimated according to the Kok model. In addition, the amplitude and the shape of the initial oxygen burst under continuous illumination are also significantly altered. At a concentration of 100 mol/l, ABA strongly inhibits Hill reaction activity measured by DCPIP reduction. The results cannot be explained by the hypothesis of socalled stomata effect. On the other hand, no effects were observed on the investigated parameters in experiments involving ABA applied in vitro to isolated chloroplasts. It is hypothesized that ABA disrupts the granal chloroplasts structure and raises the degree of participation of the cooperative mechanism of O₂-evolution connected with the functioning of PS II centers in the stroma situated thylakoids.Abbreviations DCPIP 2,6-Dichlorophenolindophenol - DCMU 3-(3,4-dichlorophenil)-1,1-dimethylurea - HEPES N-2-Hydroxyethylpiperazine-N-2-ethane sulfonic acid - PSII photosystem II - RubisCO Ribulose-1,5-bis-phosphate carboxylase-oxygenase     

Mn2+ reduces Yz + in manganese-depleted Photosystem II preparations

Manganese in the oxygen-evolving complex is a physiological electron donor to Photosystem II. PS II depleted of manganese may oxidize exogenous reductants including benzidine and Mn²⁺. Using flash photolysis with electron spin resonance detection, we examined the room-temperature reaction kinetics of these reductants with Y_z ⁺, the tyrosine radical formed in PS II membranes under illumination. Kinetics were measured with membranes that did or did not contain the 33 kDa extrinsic polypeptide of PS II, whose presence had no effect on the reaction kinetics with either reductant. The rate of Y_z ⁺ reduction by benzidine was a linear function of benzidine concentration. The rate of Y_z ⁺ reduction by Mn²⁺ at pH 6 increased linearly at low Mn²⁺ concentrations and reached a maximum at the Mn²⁺ concentrations equal to several times the reaction center concentration. The rate was inhibited by K⁺, Ca²⁺ and Mg²⁺. These data are described by a model in which negative charge on the membrane causes a local increase in the cation concentration. The rate of Y_z ⁺ reduction at pH 7.5 was biphasic with a fast 400 s phase that suggests binding of Mn²⁺ near Y_z ⁺ at a site that may be one of the native manganese binding sites.Abbreviations PS II Photosystem II - Y_D tyrosine residue in Photosystem II that gives rise to the stable Signal II EPR spectrum - Y_z tyrosine residue in Photosystem II that mediates electron transfer between the reaction center chlorophyll and the site of water oxidation - ESR electron spin resonance - DPC diphenylcarbazide - DCIP dichlorophenolindophenol