Self-Nonself Recognition Activity Extracted from Self-Sterile Eggs of the Ascidian, Ciona intestinalis

Eggs of the hermaphrodite, self-sterile ascidian, Ciona intestinalis , were washed with acid seawater (pH 3.2), and the washing solution was then adjusted to pH 8.2. This solution was found to inhibit only the binding of non-autologous sperm to the vitelline coat (VC) of eggs, indicating that it contained self-nonself recognition activity. This activity was heat-stable and insensitive to trypsin, but was destroyed by V-8 protease and α-glucosidase. Both the hydrophobic and hydrophilic components of a lyophilized powder of the extract showed allo-recognizing activity. On TLC, the hydrophobic components gave a major spot of glucose (Glc) and a peptide spot(s) containing mainly glutamic acid and/or glutamine (Glx). The glucosyl conjugate was purified by HPLC and shown to block sperm-egg binding to various extents. Individual peptide subfractions had no inhibitory activity, but in combination they showed inhibitory activity. These findings suggest that the acid extract of Ciona eggs contains a Glc-enriched nonspecific inhibitor of sperm-egg binding, which could be the primary effector of self-incompatibility, and Glx-enriched modulators, which serve as acceptors of allo-sperm. The cooperative interactions of these components may be responsible for the diversity of allo-recognition in Ciona gametes.     

Phosphoramidon inhibits the generation of endothelin-1 from exogenously applied big endothelin-1 in cultured vascular endothelial cells and smooth muscle cells

When cultured porcine aortic endothelial cells (ECs) were incubated with porcine big endothelin-1 (bit ET-1(1-39)), there was a time-dependent increase in immunoreactive (IR)-ET in the culture supernatant, in addition to an endogenous IR-ET release fron the cells. Reverse-phase HPLC of the culture supernatant revealed one major IR-ET component corresponding to the elution position of synthetic ET-1, thereby indicating that the additional increase in IR-ET was due to the conversion of big ET-1 to mature ET-1(1-21). Phosphoramidon, a metalloproteinase inhibitor, strongly suppressed this increase in IR-ET as well as the endogenous IR-ET release. Cultured vascular smooth muscle cells (VSMCs) also released IR-ET. The apparent conversion of exogenously applied big ET-1 to ET-1 and its inhibition by phosphoramidon were observed using cultured VSMCs, although the enzyme inhibitor did not influence the basal secretion of IR-ET from VSMCs. These results suggest that both cultured ECs and VSMCs can generate ET-1 from exogenously applied big ET-1 via action of the same type of phosphoramidon-sensitive metalloproteinase, which is also involved in the endogenous ET-1 generation in ECs.     

Proteasome (multicatalytic proteinase) of sea urchin sperm and its possible participation in the acrosome reaction

The egg jelly-induced acrosome reaction of the sea urchin, Strongylocentrotus intermedius, was inhibited by succinyl-Leu-Leu-Val-Tyr-4-methyl-coumaryl-7-amide (Suc-Leu-Leu-Val-Tyr-MCA), but not by Suc-Ala-Ala-Pro-Phe-MCA. The proteases with hydrolytic activity toward the former were purified from sperm extract by DEAE-Sephacel and hydroxylapatite chromatographies, Sephacryl S-300 gel filtration, and heparin-Sepharose CL-6B chromatography. Two types of protease were separated, and the molecular weights were estimated to be 65 and 700 kDa, respectively, by gel filtration. The former was accompanied by hydrolytic activity toward Suc-Ala-Ala-Pro-Phe-MCA, which was not hydrolyzed by the latter. Polyacrylamide gel electrophoresis of 700 kDa protease gave a single protein band under nondenaturing conditions and at least eight bands in the range of 22-33 kDa in the presence of sodium dodecyl sulfate (SDS). The substrate specificity and the inhibitor sensitivity of 700 kDa protease indicate that it contains two types of the activity, one is chymotrypsin-type and the other trypsin-type. The former activity was enhanced by poly-L-lysine or SDS. These properties of 700 kDa protease are similar to those of proteasomes (multicatalytic proteinases) isolated from various eukaryotic sources. We had previously shown that inhibitors of chymotrypsin-like proteases inhibit the increase of intracellular Ca2+ concentration by egg jelly, resulting in the inhibition of the acrosome reaction of St. intermedius (Matsumura and Aketa, Gamete Res 23:255-266, 1989). Bringing these findings together, we suggest that the chymotrypsin-like activity of sperm proteasome participates in the onset of the acrosome reaction of St. intermedius.     

Mechanism of induction of cellular DNA synthesis by the adenovirus E1A 12S cDNA product

The mechanism of induction of DNA synthesis in quiescent rat 3Y1 cells by the adenovirus E1A gene was investigated using the 3Y1 derivative cell lines g12-21, gn12RB1, and gn12RB2. The g12-21 cells express the E1A 12S cDNA and the latter two cells express both the E1A 12S cDNA and the human retinoblastoma susceptibility (Rb) gene at different levels in response to dexamethasone (dex). The cDNA sequences of E1A-inducible cell cycle-dependent genes, clone 3 and clone 16, were isolated by differential screening of a cDNA library constructed from dex-treated g12-21 cells. The quiescent 3Y1 cells induced c-fos and c-myc expression within 2 h after serum stimulation and expressed clone 16 and clone 3 transiently at around 8 h before the onset of DNA synthesis (10 h). In contrast, the quiescent g12-21 cells treated with dex expressed a high level of E1A at 6 to 8 h after treatment and expressed clone 16 and clone 3 at around 8 h without stimulation of c-fos and c-myc expression, suggesting that E1A bypasses the cell cycle early in G1. The half-maximal rate of DNA synthesis was reached in a much shorter time in dex-treated g12-21 cells (12 h) than in serum-treated 3Y1 cells (18 h), suggesting that E1A also bypasses the cell cycle at the G1/S boundary. The gn12RB1 and gn12RB2 cells were unable to induce DNA synthesis in response to dex presumably due to lower levels of E1A expression, although gn12RB2 but not gn12RB1 cells could express clone 16 and clone 3. These results suggest that the level of E1A required for bypass at the G1/S boundary is higher than that required early in G1.     

Crystallization of murine major histocompatibility complex class I H-2Kb with single peptides.

X-ray quality crystals of a soluble murine class I H-2Kb molecule complexed with three different peptide antigens were grown in several forms by streak seeding and macroseeding methods. Co-crystals with VSV-8 (RGYVYGQL), OVA-8 (SIINFEKL) and SEV-9 (FAPGNYPAL) peptides were grown either from NaH2PO4/HPO4 or from polyethylene glycol 4000 within the pH range 5.0 to 7.5, with the use of 4-methyl-2-pentane diol (MPD) as an additive. The VSV-8 crystals grew in space groups P1, with cell dimensions a = 63.1 A, b = 69.1 A, c = 72.0 A, alpha = 89.9 degrees, beta = 77.1 degrees, gamma = 123.3 degrees and P2(1)2(1)2, with a = 138.1 A, b = 88.6 A, c = 45.7 A, and diffract to 2.9 and 2.3 A, respectively. Crystals of the SEV-9 complex grew from similar crystallization conditions to those of the orthorhombic VSV-8 complex with similar cell parameters and diffract to at least 2.5 A resolution. Crystals of the OVA-8 complex were obtained from either phosphate (space group C2, a = 118.7 A, b = 61.6 A, c = 85.3 A, beta = 108.4 degrees) or polyethylene glycol (space group P1, a = 64.5 A, b = 71.0 A, c = 66.3 A, alpha = 89.7 degrees, beta = 95.7 degrees, gamma = 123.3 degrees) and diffract to 3 A resolution. The crystallization procedures used here significantly increased the rate and production of X-ray quality crystals.     

Continuous butanol/isopropanol fermentation in down-flow column reactor coupled with pervaporation using supported liquid membrane

Continuous butanol/isopropanol fermentation with immobilized Clostridium isopropylicum was performed in a downflow column reactor using molasses as the substrate. In order to prevent product inhibition and at the same time obtain high concentration of the products, the column reactor was coupled with a pervaporation module using a supported liquid membrane. The liquid membrane was prepared with oleyl alcohol nontoxic to the microorganism. In comparison with the continuous fermentation without product removal, the specific butanol production rate was 2 times higher. The butanol concentration in the permeate was 230 kg/m(3), which was about 50 times higher than that in the culture broth. A numerical investigation suggested a further increase in the productivity by improving the module construction.     

L-lactate production from xylose employingLactococcus lactis IO-1

Summary The growth, substrate utilisation and L-lactate production ofLactococcus lactis IO-1 were examined on xylose, and glucose and xylose media. The yield of lactate on xylose was 0.47 g lactate/g xylose at an initial xylose concentration of 51.2 g/l and the _max was 0.72 h^–1. Xylose cultures were more susceptible to lactate inhibition than were glucose cultures but showed similar kinetic behaviour. The organism was capable of complete sugar utilisation when grown on a mixture of 20 g/l xylose and 20 g/l glucose and synthesised 0.66 g lactate/g sugar.     

Phosphoramidon inhibits the conversion of intracisternally administered big endothelin-1 to endothelin-1

It is suggested that endothelin-1 (ET-1), a potent vasoconstrictor peptide, is involved in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH). We examined the effects of intracisternal administration of big ET-1 on the cerebral arteries in the absence or presence of pretreatment with phosphoramidon, an inhibitor of ET converting enzyme, in anesthetized dogs. After intracisternal administration of big ET-1 (10 micrograms/dog), the caliber of the basilar artery on the angiogram was decreased to about 59% of the control. This was accompanied by a marked increase in immunoreactive ET in the cerebrospinal fluid. Systemic arterial pressure was markedly elevated following big ET-1 injection. All changes induced by big ET-1 were effectively prevented with phosphoramidon. These data suggest that intracisternally administered big ET-1 is converted to ET-1 and that the generated ET-1 produces cerebral vasospasm and hypertension. A phosphoramidon-sensitive metalloproteinase appears to contribute to this conversion.     

A simple method forin situ freezing of anchorage-dependent cells including rat liver parenchymal cells

We developed a simple method for freezing anchorage-dependent cells, including primary cultured rat liver parenchymal cells, without detaching the cells from the culture dish. The method consists of preculture of the cells to confluence, changing the growth medium to a conventional freezing medium, packaging in a container, and storage at –80°C. After thawing and changing the freezing medium to regular growth medium, cell growth was nearly identical to that of cells freshly seeded into a new dish.     

Conversion of big endothelin-1 to endothelin-1 by two types of metalloproteinases derived from porcine aortic endothelial cells

Incubation of big endothelin-1 (big ET-1(1-39] with either the cytosolic or membrane fraction obtained from cultured endothelial cells, resulted in an increase in immunoreactive-endothelin (IR-ET), which was markedly inhibited by metal chelators. Phosphoramidon, a metalloproteinase inhibitor, specifically suppressed the membrane fraction-induced increase in IR-ET, whereas the increase in IR-ET observed with the cytosolic fraction was not influenced by phosphoramidon. Reverse-phase (RP)-HPLC of the incubation mixture of big ET-1 with the cytosolic or membrane fraction revealed one major IR-ET component corresponding to the elution position of synthetic ET-1(1-21). Simultaneously, immunoreactivities like the C-terminal fragment (CTF22-39) of big ET-1 were present, as deduced from the RP-HPLC coupled with the radioimmunoassay for CTF. Our results indicate the presence of two types of metalloproteinases, which convert big ET-1 to ET-1 via a single cleavage between Trp21 and Val22, in vascular endothelial cells.