Natriuretic activity of digoxin-like immunoreactive substance extracted from cord blood

It was examined whether the digoxin-like immunoreactive substance (DLIS) extracted from cord blood has a natriuretic activity. The DLIS was prepared from cord blood of healthy fullterm infants by acetone-HCl extraction and a gel filtration column. A solution (solution A) containing 1.0 ng/ml of DLIS or another solution (solution B) consisting of solution A from which the DLIS had been completely absorbed by rat brain synaptosome, a crude digoxin receptor, were infused directly into the renal arteries of rats. Serum and urine were serially sampled. The excretion of sodium into the urine increased gradually after the initiation of infusion and reached a level two or three times higher than that before infusion (p less than 0.05). The infusion of a buffer solution or of the extract from which the DLIS had been absorbed by rat brain synaptosome did not significantly increase the urinary excretion of sodium. Statistical analysis showed a clear difference in the natriuretic activity between solutions A and B (p less than 0.01, p less than 0.05). Well-known natriuretic substances such as atrial natriuretic hormone, prostaglandin E2, F2 alpha, bradykinin and oxytocin dopamine were not detected enough to contribute to natriuresis in the extracts. From this data, we speculated that the DLIS in cord blood has a natriuretic activity and that it plays a role in water and sodium homeostasis in perinatal life.     

Photodynamic processes and the synthesis of 5-aminolevulinic acid in chlorella cells treated with amino acids and 1,10-phenanthroline

Treatment of chlorella (Chlorella sp.) cells for 2 h in darkness with tetrapyrrole-dependent photodynamic herbicides (TDPH) derived on the basis of 0.3 mM 1,10-phenanthroline (Ph) combined with 0.6 mM Glu or 0.6 mM Gln induced the accumulation of sensitizers of photodynamic processes: magnesium protoporphyrin IX (MgPP) and MgPP monomethyl ester (MgPPE). Within the first day after chlorella cells treated with TDPH were illuminated, photodestruction of MgPP(E) was observed, and production of the first specific precursor of chlorophyll (Chl), 5-aminolevulinic acid (ALA), in the cells declined. Then the accumulation of ALA was stimulated, and the level of heme, which is a retroinhibitor of ALA synthesis, simultaneously fell. During the first two days of illumination, the content of Chl and carotenoids in the algae treated with TDPH did not differ from their levels in control culture, which suggests a high resistance of photosynthetic pigments to photodynamic process induced by porphyrins. Subsequently, a slight but rising in time accumulation of pheophytin (Pheo) was observed, as well as photodestruction of Chl and carotenoids. After five days of illumination, the difference in the content of Chl between the culture treated with TDPH and control material was 10–30% depending on the illuminance. Chlorella cells treated with TDPH remained capable of producing Chl from exogenous ALA in the dark for at least eight days. In the experiments simultaneously conducted with a higher plant, cucumber (Cucumis sativa L.), which accumulated in the dark essentially the same content of porphyrins in response to TDPH as algae did, the residual level of Chl after five days of illumination was only 10–20% of control plants. It was assumed that a high tolerance of the chlorella pigment pool to photooxidative stress induced by the accumulation of MgPP(E) and Pheo depended on a highly active state of the antioxidant protective system and the ability of ALA molecules additionally formed under the influence of TDPH to be converted into Chl, thereby participating in its de novo synthesis.     

Antiviral substance from silkworm faeces: characterization of its antiviral activity

The antiviral activity of a substance (L4-1) purified from silkworm faeces was examined in an HVJ (Sendai virus)-LLC-MK2 cell system. Its antiviral effect depended on the period of light irradiation and was inhibited by sodium sulfite and anaerobic conditions. These results indicate that the antiviral activity of L4-1 is associated with active oxygen species produced from the substance. SDS-polyacrylamide gel electrophoretic analysis showed that viral proteins were damaged by this substance under light irradiation. The results suggest that the antiviral activity is due to damage to viral protein(s) caused by active oxygen species produced from L4-1.     

The origin of nascent single-stranded DNA extracted from mammalian cells.

In vitro cultured bovine liver cells were labelled with radioactive thymidine and dissolved in 0.5% sodium dodecyl sulphate. Centrifugation of the lysate through sucrose gradients in a zonal rotor revealed a slowly sedimenting fraction of preferentially pulse labelled DNA. The DNA of this zone was further analysed by chromatography on hydroxy-apatite, banding in CsCl density gradients, and sedimentation in neutral and alkaline sucrose gradients. It contained besides small amounts of fragmented bulk DNA, single-stranded nascent DNA and single-stranded pre-labelled DNA which could be separated from each other by using BrdU as a density label. The density labelling also revealed small amounts of nascent-nascent DNA duplexes. The slowly sedimenting fraction was practically absent from cell lysates which were prepared in 2 M NaCl - 50 microgram/ml pronase. The results suggest that nascent single-strands and nascent-nascent duplexes are released from the forks of replicating DNA by branch migration. Pre-labelled single strands may be released by the same branch migration. Pre-labelled single strands may be released by the same mechanism, but the in vivo structure from which they originate has yet to be elucidated.     

Leaderless polypeptides efficiently extracted from whole cells by osmotic shock.

Three molecular foldases, DsbA, DsbC, and rotamase (ppiA), exhibited the unusual property of accumulating in an osmotically sensitive cellular compartment of Escherichia coli when their signal sequences were precisely removed by mutation. A mammalian protein, interleukin-1 (IL-1) receptor antagonist, behaved in a similar fashion in E. coli when its native signal sequence was deleted. These leaderless mutants (but not two control proteins overexpressed in the same system) were quantitatively extractable from whole cells by a variety of methods generally employed in the recovery of periplasmic proteins. A series of biochemical and genetic experiments showed that (i) leaderless DsbA (but not the wild type) was retained in a nonperiplasmic location; (ii) beta-galactosidase fusions to leaderless DsbA (but not to the wild type) exhibited efficient alpha complementation; (iii) none of the leaderless mutant proteins were substantially associated with cell membranes, even when they were overexpressed in cells; and (iv) leaderless DsbA was not transported to an osmotically sensitive compartment via a secA- or ftsZ-dependent mechanism. The observation that these proteins transit to some privileged cellular location by a previously undescribed mechanism(s)--absent their normal mode of (signal sequence-dependent) translocation--was unexpected. DsbA, rotamase, and IL-1, whose tertiary structures are known, appear to be structurally unrelated proteins. Despite a lack of obvious homologies, these proteins apparently have a common mechanism for intracellular localization. As this (putative) bacterial mechanism efficiently recognizes proteins of mammalian origin, it must be well conserved across evolutionary boundaries.     

Atp-Dependent gold accumulation by living chlorella cells

An effect of the Au(III) energy dependent concentration has been discovered by living Chlorella cells. The process is most intensive within the alkaline interval of pH, fading away in the dark, and is suppressed in the presence of arsenate (C ≧ 1 μM), fluorides (C ≧ 0.01 mM), sodium azide (1 mM), DCCD (10 μM), 2, 4-dinitrophenol (0.1 mM). In the dark the process is stimulated by ATP (but not by ADP, or AMP). ATP also neutralizes NaN₃ effect, but not that of DNP. An energy dependent Au(III) concentration is also observed for other green, blue-green, and, red singlecell algae.     

Analysis of herpes simplex virus nucleoprotein complexes extracted from infected cells.

HEp-2 cells were infected with herpes simplex virus type 1 and labeled with [3H]thymidine and 14C-amino acids. Infected cells or nuclei prepared from them were extracted with Triton X-100 and NaCl, utilizing a method recently described, and the low-speed supernatant (extract) was partially purified by sedimentation on sucrose gradients. A nucleoprotein complex which sedimented as a wide peak around 200S was identified. The nucleoprotein complex contained viral DNA, which banded at the expected density in CsCl isopycnic gradients and was intact after measurements taken on electron microscopic photographic enlargements. The autoradiographic pattern of 14C-labeled proteins after electrophoresis showed that only a few of the virus-specific polypeptides were present in the nucleoprotein complexes, in particular, VP5, VP12, VP15.2, VP19, and VP24. Cellular histones were absent. The extracts and the nucleoprotein complexes were centrifuged to equilibrium in metrizamide density gradients without prefixation. Electron microscopic direct visualization of the nucleoprotein complexes after sucrose or metrizamide purification revealed that the proteins were preferentially associated with one end of the DNA molecule and formed large irregular terminal thickenings or capsid-like transparent shells enclosing polyglobular cores. No nucleosomes were observed on herpes simplex virus nucleoprotein complexes. The same type of complex was detected after phosphonoacetic acid addition, and grossly altered nucleocapsids were formed.     

Characterization of glutamine synthetase isoforms from chlorella

Ion-exchange chromatography of extracts derived from Chlorella sorokiniana mutant strain (oxygen resistant) yielded two separate activity peaks of glutamine synthetase (GS). GS_I and GS_II were purified 220- and 187-fold and have molecular weights of approximately 398,000 and 360,000, respectively. Both enzymes are composed of eight identical subunits with a subunit molecular weight of 47,000 for GS_I and 43,000 for GS_II. The amino acid composition, catalytic, and immunological properties for both enzymes are similar.