Effect of tunicamycin on molecular heterogeneity of colony stimulating factor in cultured mouse mammary carcinoma FM3A cells.

Granulocyte and macrophage colony stimulating factors obtained from cultured mouse mammary carcinoma FM3A cells showed heterogeneity in molecular size giving rise to a major component with an apparent molecular weight of 80,000 and a minor one with that of 35,000 on Sephadex G-200 column chromatography. In the presence of tunicamycin, a specific inhibitor of asparagine-linked glycosylation, the colony stimulating factor was produced normally and consisted of a single component with an apparent molecular weight of 30,000.These data indicate that the sugar moiety is not essential for the production or activity of colony stimulating factor and that the heterogeneity in molecular size of the colony stimulating factor mainly resulted from tunicamycin-sensitive glycosylation.     

Geometrical model for the native-state folds of proteins

We recently introduced a physical model [T.X. Hoang, A. Trovato, F. Seno, J.R. Banavar, A. Maritan, Geometry and symmetry pre-sculpt the free energy landscape of proteins. Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 7960-7964, J.R. Banavar, T.X. Hoang, A. Maritan, F. Seno, A. Trovato, A unified perspective on proteins-a physics approach. Phys. Rev., E 70 (2004) 041905] for proteins which incorporates, in an approximate manner, several key features such as the inherent anisotropy of a chain molecule, the geometrical and energetic constraints placed by the hydrogen bonds and sterics, and the role played by hydrophobicity. Within this framework, marginally compact conformations resembling the native state folds of proteins emerge as broad competing minima in the free energy landscape even for a homopolymer. Here we show how the introduction of sequence heterogeneity using a simple scheme of just two types of amino acids, hydrophobic (H) and polar (P), and sequence design allows a selected putative native fold to become the free energy minimum at low temperature. The folding transition exhibits thermodynamic cooperativity, if one neglects the degeneracy between two different low energy conformations sharing the same fold topology.     

Swimming Behaviour of <Emphasis Type="Italic">Chironomus acerbiphilus</Emphasis> Larvae in Lake Katanuma

We conducted a seasonal survey of the swimming behaviour of Chironomus acerbiphilus larvae in volcanic Lake Katanuma from April 1998 to December 2001. Swimming C. acerbiphilus density was much higher than other chironomid species in lakes. All C. acerbiphilus larvae (1st through 4th instars) swam, but the earlier instars (especially the 1st) had the greatest densities and fluctuations. First instars were never found in the benthic population. This result indicates that the 1st-instar larvae are planktonic. Low water temperature (below about 10 °C) resulted in the seasonal disappearance of swimming chironomid larvae. Chemical factors – oxygen depletion or presence of hydrogen sulfide – also restricted the distribution of swimming and benthic larvae. Larvae were distributed only in the oxygen-rich part of the lake bottom and swam only in the oxygen-rich layer of the water column. The density of older swimming C. acerbiphilus (3rd and 4th instars) tended to increase with increasing benthic larval densities. The chemical stress of oxygen depletion or presence of hydrogen sulfide during holomixis within and after the stratification period leads to conspicuous swimming behaviour of benthic C. acerbiphilus larvae. Almost all C. acerbiphilus larvae died on this occasion.     

L-3-(3,4-Dihydroxyphenyl)alanine (<Emphasis Type="SmallCaps">L</Emphasis>-DOPA), an allelochemical exuded from velvetbean (<Emphasis Type="Italic">Mucuna pruriens</Emphasis>) roots

We investigated whether or not lettuce growth was inhibited by diffused L-3-(3,4-dihydroxyphenyl)alanine (L-DOPA), an allelochemical exuded from the roots of velvetbean (Mucuna pruriens (L.) DC. var. utilis) cultivars using a modified plant-box bioassay. For all the cultivars and one accession examined L-DOPA diffused from the roots and caused radicle and hypocotyl growth inhibition. A high correlation co-efficient (r = 0.838 to 0.982) was observed between L-DOPA concentration and lettuce seed sowing distance. L-DOPA diffused equally in all directions from roots at 0 mm position (close to root surface) in the plant-box, while the inhibition (%) of lettuce radicle growth gradually decreased with distance from the roots. For all cultivars the concentration of L-DOPA was significantly different at 0 mm position: being highest in cv. preta (167 g/ml) and lowest in cv. jaspeada and cv. ana (13 g/ml). The correlation between lettuce radicle growth inhibition and concentration of diffused L-DOPA was high (r = 0.856 to 0.966) in all cultivars and accession examined. However, the concentration of diffused L-DOPA did not correlate with the fresh weight concentration of L-DOPA measured in roots. The lettuce radicle growth inhibition from mucuna diffused L-DOPA was very similar that induced by synthetic L-DOPA, suggesting that diffused L-DOPA was the allelochemical responsible for growth inhibition.     

2D7 diabody bound to the alpha2 domain of HLA class I efficiently induces caspase-independent cell death against malignant and activated lymphoid cells

A mouse monoclonal antibody (2D7 mAb), which specifically bound to the alpha2 domain of HLA class I, rapidly induces cell aggregation accompanied by weak cytotoxicity against ARH-77 cells, suggesting that 2D7 mAb had a potential for agonist antibody. In order to enhance this cytotoxicity, 2D7 mAb was engineered to be a small bivalent antibody fragment, 2D7 diabody. The resultant 2D7 diabody showed a strong cytotoxicity against ARH-77 cells. As a notable characteristic feature, the lethal effect of 2D7 diabody was quite rapid, mediated by a caspase-independent death pathway. Furthermore, 2D7 diabody also showed cytotoxicity against several leukemia and lymphoma cell lines, and mitogen-activated peripheral blood mononuclear cells (PBMC), but not for normal resting PBMC and adherent cell lines such as HUVEC. These results suggest that 2D7 diabody could be expected as a novel therapeutic antibody for hematological malignancies as well as inflammatory diseases.     

Urinary neopterin quantification by reverse-phase high-performance liquid chromatography with ultraviolet detection

Neopterin plays an important role in the malignant disease diagnostics. However, the methods employed in neopterin determination are generally difficult and/or time consuming. The aim of this work was to standardize a practical method to quantify neopterin using high-performance liquid chromatography-ultraviolet (HPLC-UV) and quantify it in patients with systemic lupus erythematosus (SLE). Urine was collected from healthy subjects (n= 49), patients with inactive (n= 15), active (n= 28), and highly active SLE (n= 6). The HPLC was performed using two coupled reverse-phase columns eluted with 150 mM sodium phosphate, pH 4.0, under a flow rate of 0.8 ml/min, with UV detector set at 353 nm and 100-fold diluted urines. The inter- and intra-assay studies presented an imprecision of 12.5% and 12.9% for quality controls of 3.94 and 1.1 micromol/ml, respectively. Recovery from 79.5% to 82% was observed throughout the assay's linear range. Subjects with active (874.2 +/- 165.38 micromol/mol creatinin) and highly active SLE (1753.8 +/- 453.9 micromol/mol creatinin) showed three- and sixfold increased neopterin levels, respectively, compared to subjects with inactive SLE (314.3 +/- 121.3 micromol/mol creatinin) and healthy subjects (294.6 +/- 178.6 micromol/mol creatinin) (P< 0.05). Briefly, the proposed method was precise, specific, and reproducible, not invasive and allows the urinary neopterin quantification only with UV detection.     

Involvement of the chloroplast signal recognition particle cpSRP43 in acclimation to conditions promoting photooxidative stress in Arabidopsis

In this study, we have investigated the role of the CAO gene (coding for the chloroplast recognition particle cpSRP43) in the protection against and acclimation to environmental conditions that promote photooxidative stress. Deficiency of cpSRP43 in the Arabidopsis mutant chaos has been shown previously to lead to partial loss of a number of proteins of the photosystem II (PSII) antennae. In addition, as reported here, mutant plants have lower growth rates and reduced lignin contents under laboratory conditions. However, chaos seedlings showed significantly higher tolerance to photooxidative stress under both tightly controlled laboratory conditions and highly variable conditions in the field. This greater tolerance of chaos plants was manifested in less photooxidative damage together with faster growth recovery in young seedlings. It was also associated with a lower production of H2O2, lower ascorbate levels and less induction of ascorbate peroxidases. Under field conditions, chaos exhibited better overall photosynthetic performance and had higher survival rates. Expression of the CAO gene may be regulated by a light-dependent chloroplastic redox signalling pathway, and was inhibited during acclimation to high light and chilling temperatures, simultaneously with induction of ascorbate peroxidases. It is concluded that the presence/absence of the CAO gene has an impact on photo-produced H2O2, lignification in the hypocotyls and on the plant's susceptibility to photooxidative stress. Therefore, regulation of the CAO gene may be part of the plant's system for acclimation to high light and chilling temperatures.     

Response of the Sleep-Wake Rhythm to an 8-Hour Advance of the Light-Dark Cycle in the Rat

We have studied the effects of an 8-h advance of the environmental light-dark (LD) cycle on the sleep-wake rhythm in the rat. Electroencephalograms and electromyograms were recorded simultaneously on chart paper through a two-channel telemetry system for 3 days before phase shift (baseline) and 8 days during and after phase shift. Phase advance of the LD cycle led to an increase in both non-rapid eye movement (NREM) and REM sleep. The amount of NREM sleep in the light period correlated positively with that in the preceding dark period for 4 days after phase advance. The duration of REM sleep in the light period correlated negatively with that in the preceding dark period. The results suggest that homeostatic control of the amount of NREM sleep between the preceding dark period and the following light period is disturbed by phase advance of the LD cycle.