Activation of therminal components of human complement by a trypsin-activated complex of human factor B and cobra venom factor.

Cleavage of C3 by CVF-B was demonstrated by hemolytic, immunoelectrophoretic and immune adherence reactions. No cleavage of C5 was detected by immunoelectrophoresis, but C5 hemolytic activity, assayed with EAC1423, decreased although less than C3 hemolytic activity. The co-existence of C3 with limiting amounts of C5 did not reduce the final degree of hemolysis of guinea pig erythrocytes (GPE) induced by late-acting components C6 through C9 and CVF-B. Thus, a CVF-B hemolytic system composed of GPE, C5 through C9 and CVF-B provided a method for titration of terminal components of human complement. CVF-B was able to generate hemolytically active sites of C567 on GPE by activation of C5, C6 and C7. The complex C567 in the fluid-phase decayed within 1 min but C567 on GPE was quite stable. Originally insensitive sheep erythrocytes became sensitive to the CVF-B hemolytic system if C3b sites were present, suggesting that cell-bound C3b played a role in orienting the positions of C567 to be fixed. CVF-B could be recovered quantitatively from the supernatant of the reaction mixture in which the hemolytically active intermediate GPEC-5678 had been formed through the interaction between C5 to C8 and CVF-B.     

Synthesis and properties of mirror-image DNA.

We have investigated the conformations of the hexadeoxyribonucleotide, L-d(CGCGCG) composed of L-deoxyribose, the mirror image molecule of natural D-deoxyribose. In this paper, we report the synthesis of four L-deoxynucleosides and the L-oligonucleotide-ethidium bromide interactions. The L-deoxyribose synthon 9 was synthesized from L-arabinose with an over all yield of 28.5% via the Barton-McCombie reaction. The L-deoxynucleosides were obtained by a glycosylation of appropriate nucleobase derivatives with the 1-chloro sugar 9. After derivatization to nucleoside phosphoramidites, L-deoxycytidine and L-deoxyguanosine were incorporated into a hexadeoxynucleotide, L-d(CGCGCG) by a solid-phase beta-cyanoethylphosphoramidite method. This L-hexanucleotide was resistant to digestion with nuclease P1. The conformations of L-d(CGCGCG) were an exact mirror image of that of the corresponding natural one as described previously, and the conformations of the L-d(CGCGCG)-ethidium bromide complex were also the mirror images of those of the D-d(CGCGCG)-ethidium bromide complex under both low and high salt conditions. These results suggest that ethidium bromide prefers not a right-handed helical sense, but the base-base stacking geometry of the B-form rather than that of the Z-form. Thus, L-DNA would be a useful tool for studying DNA-drug interactions.     

Inhibition of Pisum sativum epicotyl elongation by white light – Different effects of light on the mechanical properties of cell walls in the epidermal and inner tissues

White fluorescent light (5 W m⁻²) inhibited subhook growth in derooted Alaska pea cuttings. In the inner tissue of the subhook, it inhibited the increase in osmotic potential during 18 h incubation. In the epidermis, on the other hand, light did not affect the osmotic potential. Light increased the minimum-stress relaxation time (T₀) of the inner tissue cell walls, but did not change T₀ of the epidermal cell wall. Light decreased tissue stress determined by the split test and the ability of the inner tissue to extend by water absorption. The short-term light effect on subhook growth. T₀, and the tissue stress almost disappeared when pea cuttings were transferred to darkness. These facts suggest that light changes the mechanical properties of the cell wall in the inner tissue of shoots, and decreases tissue stress, which is considered to be the driving force of shoot growth.     

Effects of environmental temperature on the population of T-cell subsets, the blastogenic responses of lymphocytes in blood and spleen and splenic NK cell activity in mice

The population of T-cell subsets, the blastogenic responses of lymphocytes in blood and spleen and splenic NK cell activity were examined in mice transferred from 22 degrees C to 12 degrees C or 32 degrees C environments. The percentage of Thy-1.2 positive cells and Lyt-1.2 positive cells in the spleen decreased after the transfer. However the percentage of Lyt-2.2 positive cells in the spleen was not affected. Thy-1.2 and Lyt-1.2 positive cells in the blood also decreased. The percentage of Lyt-2.2 positive cells in the blood was not affected in mice exposed to 12 degrees C. However, Lyt-2.2 positive cells in the blood decreased on day 1 but increased on day 3 in mice exposed to 32 degrees C. Blastogenic responses of spleen lymphocytes to concanavalin A (Con A) and pokeweed mitogen (PWM) were suppressed in transferred mice, but responses to lipopolysaccharide (LPS) and phytohemagglutinin-P (PHA-P) were not affected in any group. Blastogenic responses of blood lymphocytes to Con A, PHA-P, and PWM tended to be weaker in transferred mice than in mice kept in the 22 degrees C environment. In particular the response to PWM in mice exposed to 12 degrees C was less than 8% of that in the 22 degrees C mice. Splenic NK cell activity decreased in transferred mice, but was not suppressed as much as in mice administered 5mg of cortisone acetate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trigeminal nerve: the possible origin of substance p-nergic response in isolated rabbit iris sphincter muscle

We determined the effects of trigeminal nerve denervation on the noncholinergic, nonadrenergic response to electrical transmural stimulation of the isolated rabbit iris sphincter muscle. The left ophthalmic nerve (first branch of the trigeminal nerve) was cut at the intracranial, peripheral site of the trigeminal ganglion and five to ten days later, the iris sphincter muscle isolated from the left eye (operated side) was found to produce a fast cholinergic contraction in response to electrical transmural stimulation and there was no evidence of noncholinergic, nonadrenergic contractions. On the other hand, in the iris sphincter muscle isolated from the right eye (control side), electrical transmural stimulation produced both cholinergic and noncholinergic, nonadrenergic contractile responses. Capsaicin and bradykinin produced noncholinergic, nonadrenergic contractile responses in the muscle from the control side, while in the iris sphincter from the trigeminally denervated eye there was no such response to application of these drugs. Exogenous substance P (SP) and carbachol produced a strong contractile response in both the trigeminally innervated and denervated sphincter muscles. Somatostatin, vasoactive intestinal polypeptide (VIP) and enkephalin were without effects. These observations suggest that the noncholinergic, nonadrenergic responses to electrical transmural stimulation are derived from the trigeminal nerve and that the mediator involved is probably SP or a related peptide.     

Elimination of plasmid-linked polyglutamate production by Bacillus subtilis (natto) with acridine orange.

Treatment of Bacillus subtilis (natto) strains Asahikawa, F, and M with acridine orange resulted in the conversion of approximately 64.2% of the Asahikawa population, 22.4% of the F population, and 9.2% of the M population to polyglutamate-nonproducing colonies. Such curing is suggestive of the involvement of plasmid DNA. Samples of cleared lysates of both parental and their cured strains were subjected to agarose gel electrophoresis to determine the plasmid composition. Parental strains were found to possess a plasmid, but polyglutamate-nonproducing derivatives were missing the plasmid. The plasmid-linked polyglutamate production, which was originally isolated from B. subtilis (natto), could be transformed in B. subtilis.     

Preparation of O2-evolving Photosystem-II subchloroplasts from spinach

An O₂-evolving Photosystem II subchloroplast preparation was obtained from spinach chloroplasts, using low concentrations of digitonin and Triton X-100. The preparation showed an O₂ evolution activity equivalent to 20% of the uncoupled rate of fresh broken chloroplasts, but had no significant Photosystem-I-dependent O₂ uptake activity. The preparation showed a chlorophyll $\text{a}\text{b}$ ratio of 1.9 and a $\text{P-700}\text{chlorophyll}$ ratio of $\text{1}\text{2400}$. Absorption spectra at room temperature and fluorescence emission spectra of chlorophyll at 77 K suggested a significant decrease in Photosystem I antenna chlorophylls in the O₂-evolving Photosystem II preparation.     

Sequence analysis of the gtfC gene from Streptococcus mutans GS-5

The nucleotide sequence of the gtfC gene, which codes for glucosyltransferase synthesizing both water-soluble and water-insoluble glucans, and its flanking regions from Streptococcus mutans GS-5, was determined. Although the gtfC gene (4218 bp) is preceded by a Shine-Dalgarno (SD) sequence, a promoter-like sequence for this gene could not be identified. The gtfC gene product composed of 1375 amino acid residues (approx. 153 kDa) is generally hydrophilic with three small hydrophobic domains. Two direct repeating units were found near the C terminus of the peptide. The gtfC gene has extensive homology with the previously sequenced gtfB gene. The homologous regions correspond to the signal sequence, an internal region, and the direct repeating units of the peptide. An open reading frame preceded by an SD sequence and followed by an inverted repeat sequence was found immediately downstream from the gtfC gene. The combined sequences of the gtfB and gtfC genes as well as flanking regions suggest that the two gtf genes and the small downstream coding region could be coordinately expressed within an operon. The possible evolution of the gtfC gene in S. mutans GS-5 is also discussed.     

Calcium-activated neutral protease (CANP), a putative processing enzyme of the neuropeptide, kyotorphin, in the brain

Kyotorphin (Tyr-Arg) accumulation in the dialysed synaptosol from the rat brain in the presence of an inhibitor of kyotorphin-degrading enzyme, was maximal at neutral pH. This accumulation was activated by calcium ions, but was inhibited by leupeptin and SH-blocking agents, a finding which suggests the involvement of calcium-activated neutral protease (CANP or calpain). In addition, the kyotorphin-precursor protein, being processed by purified mu- or m-CANP, was detected at about 160 kDa on Sephacryl S-300 chromatography of the synaptosol. The present findings seem to be the first evidence for the role of CANP as a processing enzyme of neuropeptide-precursor in nerve terminals.     

Two Distinct Mechanisms Cause Heterogeneity of 16S rRNA

To investigate the frequency of heterogeneity among the multiple 16S rRNA genes within a single microorganism, we determined directly the 120-bp nucleotide sequences containing the hypervariable α region of the 16S rRNA gene from 475 Streptomyces strains. Display of the direct sequencing patterns revealed the existence of 136 heterogeneous loci among a total of 33 strains. The heterogeneous loci were detected only in the stem region designated helix 10. All of the substitutions conserved the relevant secondary structure. The 33 strains were divided into two groups: one group, including 22 strains, had less than two heterogeneous bases; the other group, including 11 strains, had five or more heterogeneous bases. The two groups were different in their combinations of heterogeneous bases. The former mainly contained transitional substitutions, and the latter was mainly composed of transversional substitutions, suggesting that at least two mechanisms, possibly misincorporation during DNA replication and horizontal gene transfer, cause rRNA heterogeneity.