Alternative complement pathway activation by C4b deposited during classical pathway activation

Sheep erythrocytes (E) sensitized with anti-E antibody (A) were reacted with guinea pig C1 (C1gp) and human C4 (C4hu) or guinea pig C4 (C4gp) to prepare EAC1, 4b. Treatment of the EAC1, 4b with a buffer containing EDTA removes C1rgp and C1sgp, resulting in the formation of EAC4b. EAC4b prepared in this way were found to be lysed by human or guinea pig serum in a gelatin Veronal-buffered saline containing 2 mM MgCl2 and 8 mM EGTA (Mg-EGTA-GVB). In the hemolytic sensitivity of EAC4bhu, essentially no difference was noted whether IgG or IgM antibodies were used for preparation of EAC4bhu. The extent of the hemolysis of EAC4bhu was dependent on the dose of C4bhu. Because EAC4bhu were lysed even by C2-deficient human serum, C3 convertase of the classical complement pathway would not be involved in the hemolysis of EAC4bhu. Furthermore, the reactivity of EAC4bhu with serum in Mg-EGTA-GVB remained even after treatment of the intermediate cells with 1 mM PMSF, indicating that any remaining C1gp was not responsible for the hemolysis. Therefore, the hemolysis of EAC4b by sera in Mg-EGTA-GVB was considered to be mediated via activation of the alternative complement pathway (ACP). Pretreatment of EAC4bhu with anti-C4hu antibody or C4-binding protein suppressed the hemolysis of EAC4bhu via the ACP activation. Furthermore, EAC4bhu were more sensitive to hemolysis by the reaction with a mixture of C3, B, D, and H followed by rat serum in EDTA-GVB than EAC1qgp were. These results indicate that C4b molecules on the cell membrane participate in the activation of ACP.     

Significant increase in plasma immunoreactive atrial natriuretic polypeptide concentration during head-out water immersion

To investigate the physiological regulatory mechanism of human atrial natriuretic polypeptide (hANP) secretion, plasma hANP was measured by a direct radioimmunoassay during head-out total body water immersion (WI) in normal men. Five healthy men were immersed in water for 1 hr. Urine volume and Na excretion were significantly increased during WI. Plasma hANP increased significantly during WI peaking at 30 min. and returned toward the baseline after WI. Plasma renin activity and norepinephrine were suppressed occasionally during WI. Plasma ADH did not change throughout the study period. Maximal increments in plasma hANP correllated with that in urine output or urinary Na excretion during WI. These data suggest that acute central hypervolemia caused by WI increases hANP secretion and that this increase may participate in the diuretic response to WI.     

Unusual lysosomes in aortic smooth muscle cells: presence in living and rapidly frozen cells

Unusual tubular structures have been observed in rat aortic smooth muscle cells (SMC) grown in culture. These tubular structures have several characteristics that strongly suggest that they are lysosomes: they are bounded by a single membrane bilayer, contain densely staining material, and acid phosphatase activity. Furthermore, these structures are present in living cells, as demonstrated by their ability to accumulate the membrane-impermeable fluorescent dye lucifer yellow CH. In ultrastructural preparations they are best seen in samples that are cryofixed by rapid freezing and then freeze-substituted in osmium-acetone solutions. Conventional chemical fixation did not appear to preserve these structures to as great an extent as did rapid freezing. Comparison of SMC in vitro to the same cells in situ revealed differences in lysosome number as well as morphological appearance. Thus, the culturing of rat SMC leads to the formation of unusual tubular lysosomes whose ultrastructural appearance is particularly sensitive to the methods employed for examination.     

Increases in cytosolic free Ca2+ induced by ATP, complement and beta-lipoprotein in mouse L fibroblasts

By means of Ca2+- and K+-selective microelectrodes, the changes in intracellular free Ca2+ and K+ were measured during the hyperpolarizing responses induced by ATP, complement and beta-lipoprotein in mouse fibroblastic L cells. The cytoplasmic Ca2+ concentration [( Ca]i) was about 0.4 microM in the resting state. The hyperpolarizing responses always coincided with a phasic increase in [Ca]i. ATP or beta-lipoprotein induced about a 2-fold rise in [Ca]i, and complement did up to 3-fold. Both the hyperpolarizing responses and [Ca]i increases were prevented by removal of external Ca2+ or by application of a Ca-channel blocker, nifedipine. Quinine, a Ca-activated K-channel inhibitor, suppressed the hyperpolarizing responses but not the [Ca]i increases. During the hyperpolarizing response, the intracellular free K+ concentration gradually decreased from about 120 to 110 mM. Thus, it is concluded that ATP, complement and beta-lipoprotein caused a transient elevation of cytoplasmic free Ca2+ due to Ca2+ influxes, thereby inducing electrical membrane responses through activation of Ca-dependent K-channels in the fibroblasts.     

Atrial natriuretic factor increases cyclic GMP and inhibits cyclic AMP in rat renal papillary collecting tubule cells in culture

The present study was undertaken to determine whether human atrial natriuretic factor (hANF) produces guanosine-3', 5'-monophosphate (cGMP) and alters arginine vasopressin (AVP)- and forskolin (F)- induced adenosine-3', 5'-monophosphate (cAMP) production in the cultured rat renal papillary collecting tubule cells. hANF increased cellular cGMP levels in a dose dependent manner. AVP and F, however, did not affect cGMP production. hANF significantly inhibited AVP- and F-stimulated cAMP levels, but hANF by itself did not affect cellular cAMP production. Since F activates adenylate cyclase at a step of catalytic unit and the cellular action of AVP to activate adenylate cyclase is mediated through receptor-catalytic units, the present results indicate that hANF may directly inhibit the AVP- and F-stimulated adenylate cyclase in renal papillary collecting tubules.     

Oxidation of acyclic terpenoids by Corynebacterium sp.

Squalene analogs such as lycopersene, geranylfarnesyl, digeranyl, and 2-hydroxy-2,3-dihydrosqualene and terpene alcohol derivatives such as farnesyl benzyl ether, farnesyl pivalate, geranylgeranyl pivalate, geranyl pivalate, and geranyl benzyl ether were oxidized by Corynebacterium sp. strain SY-79, which was isolated from soil by using squalene as a carbon source. Lycopersene and geranylfarnesyl gave no major product. Digeranyl, geranyl benzyl ether, and geranyl pivalate gave terminal oxidation products, and 2-hydroxy-2,3-dihydrosqualene, farnesyl benzyl ether, farnesyl pivalate, and geranylgeranyl pivalate were degraded to give lower molecular carboxylic acids. Strain SY-79 showed promising oxidative activities toward acyclic terpenes, although the metabolites obtained were variable, depending upon the structure of the substrate.     

Synthesis of a nonacosapeptide (beta-fragment) corresponding to the N-terminal sequence 1-29 of human liver metallothionein II and its heavy metal-binding properties

A nonacosapeptide (beta-fragment) corresponding to the N-terminal sequence 1-29 of human liver metallothionein II was synthesized by the fragment condensation method. The Cd-binding ability of the beta-fragment was much stronger than that of cysteine as thionein and synthetic alpha-fragment corresponding to the C-terminal sequence 30-61 of human liver metallothionein II. Both the alpha- and beta-fragments bound preferentially to Cu ions rather than Cd ions.     

The production of cloned fish in the medaka (Oryzias latipes)

The measurement of cellular DNA content by DNA microfluorometry revealed that medaka embryos that were fertilized with normal sperm and exposed to heat shock (41 degrees C for 3 min) or hydrostatic pressure (700 kg/cm2 for 10 min) at 85-95 min after insemination were tetraploid. Embryos fertilized with normal sperm and exposed to heat shock (41 degrees C for 2 min at 2-3 min after insemination) were triploid. These results suggest that heat shock or hydrostatic pressure at 85-95 min after insemination arrests the first cleavage, while heat shock at 2-3 min after insemination arrests the second meiotic division. Medaka clones have been produced by the following method: Eggs from orange-red or variegated variety were activated by UV-irradiated, genetically impotent sperm of wild-type fish (UV sperm). The haploid eggs obtained were diploidized by preventing the first cleavage with heat shock or hydrostatic pressure to produce homozygous females. Each of the two homozygous females was mated with vasectomized male in isotonic balanced salt solution to collect unfertilized eggs. The collected eggs were activated with UV sperm and converted from haploid to diploid by arrest of the second meiotic division with heat shock. Hatched fry of each homozygous diploid (all females) were fed with a methyltestosterone-containing diet (40 micrograms/gm diet) to produce sex-reversed males, which were mated with brood females, and thus two cloned lines were obtained.     

Purification of Ribulose 5-phosphate Kinase and Minor Polypeptides of Pyrenoid from the Green Alga Bryopsis maxima

Ribulose 5-phosphate (Ru5P) kinase (ATP:D-ribulose 5-phosphate1-phosphotrans- ferase; EC 2.7.1.19 [EC] ), an enzyme in the reductivepentose phosphate cycle, was purified from the green alga Bryopsismaxima and its activity and peptide composition were studied.The specific activity of purified Ru5P kinase was 20 µmoleRuBP formed (mg protein)^–1 min^–1 corresponding toa 490-fold purification from the supernatant of chloroplasts.The K_m values of Ru5P kinase for ATP and Ru5P were 69 µMand 330 µM, respectively. The molecular size of Ru5P kinase was estimated as 90 kDa bygel filtration and that of its polypeptide as 41 kDa by SDS-polyacrylamidegel electrophoresis. A small portion of the Ru5P kinase wasfound in a large molecular state (500 kDa) which was consideredto be an inactive form of the enzyme. Ru5P kinase activity has been reported in the pyrenoid of Eremosphaeraviridis as well as ribulose 1,5-bisphosphate carboxylase-oxygenase(RuBisCO) and ribose 5-phosphate isomerase activity (Holdsworth1971). In Bryopsis maxima, among the pyrenoid polypeptides otherthan that of RuBisCO, we found a polypeptide of 42 kDa, similarto that of Ru5P kinase in molecular size and ratio to RuBisCO.A peptide map of the 42 kDa pyrenoid polypeptide, however, showedthat it differed from that of Ru5P kinase. In conclusion, Ru5Pkinase may be not involved in the pyrenoid of this alga. (Received January 19, 1985; Accepted May 15, 1985)