Immunolocalization of Na,K-ATPase in blowfly photoreceptor cells

The Na,K-ATPase (sodium pump) plays a central role in the physiology of arthropod photoreceptors as it re-establishes gradients for Na⁺ and K⁺ after light stimulation. We have mapped the distribution of the Na,K-ATPase in the photoreceptors of the blowfly (Calliphora erythrocephala) by immunofluorescent and immunogold cytochemistry, and demonstrate that the distribution pattern is more complex than previously presumed. High levels of sodium pumps have been detected consistently in all photoreceptors R1-8 on the nonreceptive surface, but no sodium pumps are found on the microvillar rhabdomere. Within the nonreceptive surface of the cells R1-6, however, the sodium pumps are confined to sites juxtaposed to neighboring photoreceptor or glial cells; no sodium pumps have been detected on the parts of the nonreceptive surface exposed to the intra-ommatidial space. In R7 and R8, the sodium pumps are found over the entire nonreceptive surface. The cytoskeletal protein spectrin colocalizes with the sodium pumps suggesting that linkage of the pump molecules to the spectrin-based submembrane cytoskeleton contributes to the maintenance of the complex pattern of pump distribution.     

Dopamine-Releasing Action of 6R-l-erythro-Tetrahydrobiopterin: Analysis of Its Action Site Using Sepiapterin

Abstract: Recently, we reported that 6 R - l - erythro -tetrahydrobiopterin (6 R -BH₄), a natural cofactor for hydroxylases of tyrosine and tryptophan, has a monoamine-releasing action independent of its cofactor activity. Here we attempted to determine whether 6 R -BH₄ acts inside the cell or from the outside of the cell by using brain microdialysis in the rat striatum. For this purpose, sepiapterin, an immediate precursor of 6 R -BH₄ in the salvage pathway, was used to selectively increase the intracellular 6 R -BH₄ levels. Dialytic perfusion of sepiapterin increased tissue levels of reduced biopterin (mainly 6 R -BH₄) but not the extracellular levels. Administration of sepiapterin increased the extracellular levels of 3,4-dihydroxyphenylalanine (DOPA) (an index of in vivo tyrosine hydroxylase activity) and of dopamine (DA) (an index of in vivo DA release). Either of the increases was eliminated after pretreatment with a tyrosine hydroxylase inhibitor α-methyl- p -tyrosine. Administration of 6 R -BH₄ increased extracellular levels of reduced biopterin, DOPA, and DA. After pretreatment with α-methyl- p -tyrosine, the increase in DOPA levels was abolished, but most of the increase in DA levels persisted. The increase in DA levels also persisted after pretreatment with nitric oxide synthase inhibitors. These data demonstrate that 6 R -BH₄ stimulates DA release directly, independent of its cofactor action for tyrosine hydroxylase and nitric oxide synthase, by acting from the outside of neurons.     

DEVELOPMENT, SEXUAL DIMORPHISM, and INDIVIDUAL VARIATION IN THE SKELETON OF THE FINLESS PORPOISE, NEOPHOCAENA PHOCAENOIDES, IN THE COASTAL WATERS OF WESTERN KYUSHU, JAPAN

Development, sexual dimorphism, and individual variation were examined in the skeleton of the finless porpoise in the coastal waters of western Kyushu, Japan. Skulls ceased growing by 4 yr. Postcranial skeletons ceased increasing in size at an age older than 11 yr. The finless porpoise was estimated to attain cranial maturity by 4 yr and physical maturity at 14–23 yr. Sexual dimorphism was not detectable in most of the cranial characters but was detected in more than half of the postcranial characters. Females tended to show larger values of postcranial characters. The shape of the pelvic bone was obviously different between males and females. Thus, a discriminant function was proposed to determine sex using measurements of this bone. Individual variation was greatest in the feeding apparatus such as length of the rostrum, and least in the braincase.     

Inhibition of the catalase reaction of Photosystem II by anions

A new binding site for anions which inhibit the water oxidizing complex (WOC) of Photosystem II in spinach has been identified. Anions which bind to this site inhibit the flash-induced S₂/S₀ catalase reaction (2H₂O₂2H₂O+O₂) of the WOC by displacing hydrogen peroxide. Using a mass spectrometer and gas permeable membrane to detect the ³²O₂ product, the yield and lifetime of the active state of the flash-induced catalase (to be referred to simply as flash-catalase) reaction were measured after forming the S₂ or S₀-states by a short flash. The increase in flash-catalase activity with H₂O₂ concentration exhibits a K_m=10–20 mM, and originates from an increase in the lifetime by 20-fold of the active state. The increased lifetime in the presence of peroxide is ascribed to formation of the long-lived S₀-state at the expense of the unstable S₂-state. The anion inhibition site differs from the chloride site involved in stimulating the photolytic water oxidation reaction (2H₂OO₂+4e^-+4H⁺). Whereas water oxidation requires Cl^- and is inhibited with increasing effectiveness by F^-CN^-N₃ ^-, the flash-catalase reaction is weakly inhibited by Cl^-, and with increasing effectiveness by F^-CN^-, N₃ ^-. Unlike water oxidation, chloride is unable to suppress or reverse inhibition of the flash-catalase reaction caused by these anions. The inhibitor effectiveness correlates with the pK_a of the conjugate acid, suggesting that the protonated species may be the active inhibitor. The reduced activity arises from a shortening of the lifetime of the flash-induced catalase active state by 3–10 fold owing to stronger anion binding in the flash-induced states, S₂ and S₀, than in the dark S-states, S₁ and S_-1. To account for the paradoxical result that higher anion concentrations are required to inhibit at lower H₂O₂ concentrations, where S₂ forms initially after the flash, than at higher H₂O₂ concentrations, where S₀ forms initially after the flash, stronger anion binding to the S₀-state than to the S₂-state is proposed. A kinetic model is given which accounts for these equilibria with anions and H₂O₂. The rate constant for the formation/release of O₂ by reduction of S₂ in the WOC is <0.4 s^-1.Abbreviations ADRY acceleration of the deactivation reactions of the water splitting enzyme system Y - BTP bis [tris(hydroxymethyl)methylamino]-propane - CCCP carbonylcyanide m-chlorophenylhyrazone - DCBQ 2,5-dichlorobenzoquinone - DMBQ 2,3-dimethylbenzoquinone - WOC water oxidizing complex     

Secondary structural changes of large and small fragments of bovine serum albumin in thermal denaturation and in sodium dodecyl sulfate denaturation

The helicities in various fragments of bovine serum albumin (BSA) were examined in the thermal denaturation and in sodium docecyl sulfate (SDS) denaturation. The thermal denaturation was examined in a temperature range between 2 and 65°C. The helicity decreased with a rise of temperature and it recovered to some degree upon cooling temperature. A rather high reversibility was observed in the BSA fragments, which were located in the N-terminal of the parent protein and then contained the first large loop with no disulfide bridge. The high reversibility was available also for the helicity in the first large loop of the fragment, disulfide bridges of which were reduced. The fragments, which were smaller than one domain, became unstable in the SDS denaturation. The helicities of such fragments decreased in lower SDS concentrations compared with those of the intact BSA and the large fragments, which contained one or more domains. A resistance to the SDS denaturation appeared in the helices of every large loop even after the fragmentation. On the other hand, helicities of the fragments decreased to 20–25% upon the reduction of disulfide bridges. However, the helicities of these fragments increased to 35–40% in the SDS denaturation.     

cDNA cloning and mRNA expression of calponin and SM22 in rat aorta smooth muscle cells

We cloned and sequenced cDNAs encoding calponin (Calp) and SM22 (smooth muscle-specific 22-kDa protein) from rat aorta (RaA) smooth muscle (Smu) cells. The 1504-bp calp cDNA contains a single open reading frame (ORF) which encodes 297 amino acids (aa) (M_r 33 342). The 1186-bp SM22 cDNA contains a single ORF which encodes 201 aa (M_r 22 601). There were 43% identical aa in a 181-aa overlap between RaA Calp and SM22. Especially for the C-terminal region of SM22 and for the first repeat motif of Calp, 70% identity was observed. Northern blot analysis revealed that the calp and SM22 mRNAs were expressed in RaA Smu, but not in rat cardiac and skeletal muscles. SM22 mRNA was much more abundant than calp mRNA in RaA (3- to 4-fold). The expression levels of the calp and SM22 mRNAs in RaA showed a significant increase for 5 to 15 week old rats (1.5- to 3-fold) with vascular development and blood pressure elevation. No significant differences were observed in the expression of the RaA calp and SM22 mRNAs between normotensive (Wistar Kyoto) and spontaneously hypertensive rats (SHR).     

AGE, GROWTH, AND REPRODUCTION OF THE FINLESS PORPOISE, NEOPHOCAENA PHOCAENOIDES, IN THE COASTAL WATERS OF WESTERN KYUSHU, JAPAN

Abstract: In the coastal waters of western Kyushu, Japan, a total of 97 incidentally taken or stranded finless porpoises, Neophocaena phocaenoides , was collected for studying age, growth and reproduction. An additional 17 specimens from the Inland Sea were used for a comparison of life history. Mean neonatal body length was 78.2 cm. Both males and females grew to around 140 cm by 5 yr of age. The maximum body lengths of males and females in western Kyushu were 174.5 cm and 165.0 cm, respectively, which were smaller than those recorded in other Japanese waters. Females probably attain sexual maturity at ages of 6–9 yr and at body lengths of 135–145 cm. Males probably mature sexually at ages of 4–6 yr, at body lengths of 135–140 cm and at weight of testis of 40–150 g. The lack of females aged 5–6 yr and males aged 4–5 yr precluded firm conclusions on ages at sexual maturity. Parturition in western Kyushu was estimated to be prolonged from autumn to spring, whereas in the Inland Sea and Pacific waters it was restricted from spring to summer with a peak in April. These geographical differences and available information on distribution implies that the finless porpoises in western Kyushu constitute a local population.     

DIEL CHANGES IN THE COMPOSITION OF PHOTOSYNTHETIC PIGMENTS AND CELLULAR CARBON AND NITROGEN IN CHATTONELLA ANTIQUA (RAPHIDOPHYCEAE)1

An axenic clonal culture of Chattonella antiqua (Hada) Ono was grown on a 12: 12 h LD cycle in a laboratory culture tank containing 1 m³ of f/2 medium. Diel changes in mean cell volume, cellular carbon (carbon content per cell), C/N ratio, cellular Chl a, Chl a/c ratio and carotenoid composition were observed. Mean cell volume and cellular C, N and pigments increased during the light period as a result of photosynthesis and decreased with increase of cell concentration by phased cell division during the dark period. These changes indicated that carbon assimilation and pigment synthesis occurred together during the light period. However, the patterns of increase were not the same since different diel patterns were also found in the ratios of C/N and chl a/c. Photosynthetic pigments were analyzed by reversed-phase high-performance liquid chromatography with ion-pairing solution. This analysis showed that the dominant carotenoids in C. antiqua were fucoxanthin, violaxanthin and β-carotene. Diel patterns of Chls a and c were similar to that of fucoxanthin but different from those of violaxanthin and β-carotene. The cellular contents of Chl a, fucoxanthin and carbon increased in a parallel manner during the light period. On the other hand, the increase of violaxanthin was restricted to only a few hours at the beginning of the light period during cell division cycles.