Structure-function studies on the interaction of PsaC with the Photosystem 1 heterodimer

The interaction of PsaC with the core heterodimer of Photosystem 1 was studied in wild type Synechocystis sp. PCC 6803 and the site-directed mutant R561E of PsaB. The mutant reaction center was much less stable in urea and the functional reconstitution of the mutant core using PsaC was impaired. However, the extent of reconstitution increased in the presence of divalent cations whereas that of the wild type was inhibited. We conclude that the reaction center in the mutant is unstable, most likely due to the introduction of an unfavorable electrostatic interaction between surface-exposed residues on PsaC and the binding site for the subunit on the core heterodimer in support of the model proposed previously (Rodday et al. (1993) Photosynth Res 36: 1–9).Abbreviations PEG polyethylene glycol - 1 - PS1 Photosystem 1 - PMSF phenylmethylsulphonyl fluorid - TMPD N, N, N, N tetramethyl-p-phenylenediamine - TPCK N-tosyl-Phe chloromethyl ketone     

Sexual steroids during puberty in male African catfish (Clarias gariepinus): serum levels and gonadotropin-stimulated testicular secretion in vitro

Blood serum and testicular tissue samples were collected from 3 to 13-month-old African catfish (groups A-G) in order to study their pubertal development. The sampling covered the period from before the beginning of spermatogenesis until full maturity. Testes of fish in group A contained spermatogonia alone. In testes of group B, spermatogonia, spermatocytes and spermatids were present. Spermatozoa were first observed in group C and became predominant as the fish attained full maturity (group G). Several sex steroids were determined in the blood samples. Testosterone was the quantitatively dominating androgen in the blood serum (3–5 ng·ml^-1) in groups B and C (fish in group A were too small to collect blood samples). In group D, the concentrations of 11-ketotestosterone and 11-hydroxyandrostenedione increased to levels similar to those of testosterone. Androstenedione that was undetectable before (below 0.4 ng·ml serum^-1), also increased to 3–5 ng·ml^-1 in group D. The levels of androgens kept increasing until the fish attained full maturity (group G). In order to monitor the responsiveness to gonadotropic hormone and the steroid secretion capacity, the in vitro secretion of two steroids (11-hydroxyandrostenedione and 17,20-dihydroxy-4-pregnen-3-one) by testicular tissue was quantified at the different stages of development. Testicular maturation was accompanied by changes of both the steroid secretion capacities and of the sensitivity to gonadotropic hormone. The most important changes occurred just after the initiation of spermatogenesis, as spermatocyte/spermatid formation was associated with a drop of the secretory capacity (amount of steroid secreted per milligram of tissue incubated) and with a reduced sensitivity to gonadotropic hormone. At later stages, when the testicular weight substantially increased concurrently with the formation of numerous spermatozoa, both the secretory capacity and the responsiveness to gonadotropic hormone increased again to reach the levels typical of adult fish. The blood levels of androgens appeared to be positively related to the increasing testicular weight in the later phases of development.Abbreviations 17,20P 17,20-dihydroxy-4-pregnen-3-one - A2 androstenedione - A3 androstenetrione - BPG-axis brain-pituitary-gonadal axis - FSH follicle stimulating hormone - GnRH gonadotropin-releasing hormone - GTH gonadotropic hormone - GSI gonado-somatic index - hCG human chorionic gonadotropin - HEPES N-(2-hydroxyethyl)piperazine-N-(2-ethanesulphonic acid) - KT 11-ketotestosterone - LH luteinizing hormone - OHA 11-hydroxyandrostenedione - OHT 11-hydroxytestosterone - PE pituitary extract from adult fish - PE_juv pituitary extract from juvenile fish - RIA radio immunoassay - T testosterone     

Differential localization of vacuolar H+-ATPases containing a1, a2, a3, or a4 (ATP6V0A1-4) subunit isoforms along the nephron.

Vacuolar H(+)-ATPase are multi-subunit containing pumps important for several processes along the nephron such as receptor mediated endocytosis, acidification of intracellular organelles, bicarbonate reabsorption and secretion, and H(+)- extrusion. Mutations in the human a4 (ATP6V0A4) subunit cause distal renal tubular acidosis (dRTA). There are 4 known isoforms of the 'a' subunit (a1-a4). Here we investigated the expression and localization of all four isoforms in mouse kidney. Real-time PCR detected mRNAs encoding all four 'a' isoforms in mouse kidney with a relative abundance in the following order: a4>a2=a1>a3. Immunolocalization demonstrated expression of all 'a' subunits in the proximal tubule and in the intercalated cells of the collecting system. In intercalated cells a1 and a4 isoforms appeared on both the apical and basolateral side and were expressed in all subtypes of intercalated cells. In contrast, a2, and a3 were only found in the apical membrane. a1 and a4 were colocalized in the same cells with AE1 or pendrin, whereas a2 was only found in AE1 positive cells but absent from pendrin expressing intercalated cells. These results suggest that vacuolar H(+)-ATPases containing different 'a' isoforms may serve specific and distinct functions and may help explaining why loss of the a4 isoform causes only dRTA without an apparent defect in the proximal tubule.     

Glycogen, ammonia and related metabolities in the brain during seizures evoked by methionine sulphoximine

Abstract— The levels of ATP, P-creatine, glucose, glycogen, lactate, glutamate and ammonia were measured in mouse brain after administration of the convulsive agent methionine sulphoximine (MSO). No changes were observed in ATP and P-creatine levels either before or during the seizures. Lactate levels were unchanged until the onset of seizures (4–5 hr) at which time the levels increased an average of 65 per cent. Glucose and glycogen levels increased progressively. Just before the onset of seizures the levels had increased 95 and 62 per cent, respectively. During the seizures both substances had increased a total of 130 per cent. Comparable changes were found in cerebral cortex, cerebellum and subcortical forebrain. Through the use of quantitative histochemical methods it was found that the greatest increases in glycogen occurred in layers I and III (layers II and IV were not analysed). Progressively smaller changes were found in layers V and VI and no increase at all was found in the subjacent white matter. Glucose, in contrast to glycogen, increased to about the same degree in all cerebral layers and in subjacent white matter. The increase in glycogen after MSO administration may be related to the fact that MSO also causes an increase in the ratio of brain to serum glucose levels. This would indicate that an increase in intracellular glucose had occurred. Ammonia levels were increased 300–400 per cent in both cerebrum and cerebellum. A time study in cerebellum showed that the increase begins early and reaches maximal levels long before the onset of seizures. Glutamate levels were reduced by small but statistically significant amounts in both cerebrum and cerebellum. Administration of methionine sulphoximine completely prevented seizures and the increase in lactate, but did not prevent the increases in glycogen and glucose. The rise in ammonia was reduced but not prevented. During 20 sec of complete ischaemia (decapitation) ATP, P-creatine and glucose fell somewhat more rapidly than normal in brain of animals undergoing MSO seizures. From the changes it was calculated that the metabolic rate had been increased about 20 per cent by the seizure. A new sensitive and specific enzymic method for determination of tissue ammonia is presented together with evised enzymic procedures for lactate and glutamate.     

EFFECTS OF VISIBLE AND ULTRAVIOLET RADIATION ON THE GERMINATION OF PHACELIA TANACETIFOLIA

Schulz , Sister M. Richardis , O.P., and Richard M. Klein . (N. Y. Bot. Gard., N. Y., N. Y.) Effects of visible and ultraviolet radiation on the germination of Phacelia tanacetifolia. Amer. Jour. Bot. 50(5): 430–434. Illus. 1963.—Germination of Phacelia tanacetifolia was suppressed by exposure to white light increasing with intensity and length of illumination. The light effect decayed during 24 hr of darkness. Seeds were most sensitive to the suppressive effects of light 13–17 hr after the beginning of imbibition. Light suppression was caused by a photocatalytic reaction. Wavelengths causing the suppression lie in the far-red, red, blue, near-ultraviolet and far-ultraviolet regions of the spectrum. At equal energies, blue light was less effective than far-red, red or ultraviolet radiation. There was no evidence for the existence of the phytochrome system. Simultaneous irradiation with red and blue light or simultaneous irradiation with red and far-red induced a synergistic repression of germination. The presentation of different wavelengths in various sequential patterns markedly altered the germination response. An interaction between elevated temperatures and visible radiation affecting germination response was also noted.     

Protein–protein interactions indicate composition of a 480 kDa SELMA complex in the second outermost membrane of diatom complex plastids

Most secondary plastids of red algal origin are surrounded by four membranes and nucleus‐encoded plastid proteins have to traverse these barriers. Translocation across the second outermost plastid membrane, the periplastidal membrane (PPM), is facilitated by a ERAD‐(ER‐associated degradation) derived machinery termed SELMA (symbiont‐specific ERAD‐like machinery). In the last years, important subunits of this translocator have been identified, which clearly imply compositional similarities between SELMA and ERAD. Here we investigated, via protein–protein interaction studies, if the composition of SELMA is comparable to the known ERAD complex. As a result, our data suggest that the membrane proteins of SELMA, the derlin proteins, are linked to the soluble sCdc48 complex via the UBX protein sUBX. This is similar to the ERAD machinery whereas the additional SELMA components, sPUB und a second Cdc48 copy might indicate the influence of functional constraints in developing a translocation machinery from ERAD‐related factors. In addition, we show for the first time that a rhomboid protease is a central interaction partner of the membrane proteins of the SELMA system in complex plastids.