Variation of loop sequence alters stability of cytolethal distending toxin (CDT): crystal structure of CDT from Actinobacillus actinomycetemcomitans

Cytolethal distending toxin (CDT) secreted by Actinobacillus actinomycetemcomitans induces cell cycle arrest of cultured cells in the G2 phase. The crystal structure of the natural form of A. actinomycetemcomitans DCT (aCDT) has been determined at 2.4 A resolution. aCDT is a heterotrimer consisting of the three subunits, aCdtA, aCdtB, and aCdtC. Two crystallographically independent aCDTs form a dimer through interactions of the aCdtB subunits. The primary structure of aCDT has 94.3% identity with that of Haemophilus ducreyi CDT (hCDT), and the structure of aCDT is quite similar to that of hCDT reconstituted from the three subunits determined recently. However, the molecular packings in the crystal structures of aCDT and hCDT are quite different. A careful analysis of molecular packing suggests that variation of the amino acid residues in a nonconserved loop (181TSSPSSPERRGY192 of aCdtB and 181NSSSSPPERRVY192 of hCdtB) is responsible for the different oligomerization of very similar CDTs. The loop of aCdtB has a conformation to form a dimer, while the loop conformation of hCdtB prevents hCDT from forming a dimer. Although dimerization of aCDT does not affect toxic activity, it changes the stability of protein. aCDT rapidly aggregates and loses toxic activity in the absence of sucrose in a buffered solution, while hCDT is stable and retains toxic activity. Another analysis of crystal structures of aCDT and hCDT suggests that the receptor contact area is in the deep groove between CdtA and CdtC, and the characteristic "aromatic patch" on CdtA.     

The effect of Cyclin‐dependent kinase 5 on voltage‐dependent calcium channels in PC12 cells varies according to channel type and cell differentiation state

Cyclin‐dependent kinase 5 (Cdk5) is a Ser/Thr kinase that plays an important role in the release of neurotransmitter from pre‐synaptic terminals triggered by Ca²⁺ influx into the pre‐synaptic cytoplasm through voltage‐dependent Ca²⁺ channels (VDCCs). It is reported that Cdk5 regulates L‐, P/Q‐, or N‐type VDCC, but there is conflicting data as to the effect of Cdk5 on VDCC activity. To clarify the mechanisms involved, we examined the role of Cdk5 in regulating the Ca²⁺‐channel property of VDCCs, using PC12 cells expressing endogenous, functional L‐, P/Q‐, and N‐type VDCCs. The Ca²⁺ influx, induced by membrane depolarization with high K⁺, was monitored with a fluorescent Ca²⁺ indicator protein in both undifferentiated and nerve growth factor (NGF)‐differentiated PC12 cells. Overall, Ca²⁺ influx was increased by expression of Cdk5‐p35 in undifferentiated PC12 cells but suppressed in differentiated PC12 cells. Moreover, we found that different VDCCs are distinctly regulated by Cdk5‐p35 depending on the differentiation states of PC12 cells. These results indicate that Cdk5‐p35 regulates L‐, P/Q‐, or N‐type VDCCs in a cellular context‐dependent manner.

     

Production of high levels of interleukin 1-like activity by the SPI-802 human leukemia cell line with E receptors

The SPI-802 human leukemia cell line, which possesses E receptors and used to have natural killer activity, has been demonstrated to produce high levels of interleukin 1 (IL-1)-like activity. SPI-802 supernatants prepared in 1% serum-containing cultures with lipopolysaccharide stimulation, like similarly prepared adherent-cell-derived IL-1, enhanced phytohemagglutinininduced mouse thymocyte proliferation. When adherent-cell IL-1 gave 50% maximum activity at a reciprocal dilution of 20, SPI-802 supernatant gave it at 200, indicating the production of high levels of IL-1-like activity by the cell line. SPI-802 supernatant promoted the production of interleukin 2 (IL-2) by the Jurkat-F1884 T-cell line: Levels of IL-2 activity obtained with 15% SPI-802 supernatant were almost equivalent to those obtained with 50% adherent-cell IL-1 as estimated by the maximum proliferation of IL-2-dependent cytotoxic T cells. SPI-802 supernatant by itself exhibited no IL-2 activity. Major IL-1-like activity of SPI-802 supernatant was present in fractions from AcA54 columns corresponding to M, 12,000–20,000 and 60,000–70,000 and resolved on isoelectrofocusing into two distinct species with pI values of 5.0 and 7.0, being consistent with the results of adherent-cell IL-1. The SPI-802 cell line having E receptors is an ideal source of a soluble factor with the biological and biochemical Characteristics of human IL-1.     

Haemolytic-uraemic syndrome complicating shigella dystentery in south Indian children.

Shigella dysentery caused 65% of all cases of acute renal failure (ARF) seen in children treated at the Christian Medical College Hospital, Vellore, during the 33 months ending September 1977. In the 40 children with ARF secondary to shigella dysentery, haematological findings suggested that they were suffering from the haemolytic-uraemic syndrome, and glomerular hypercellularity and fibrin deposition were present in all 12 patients whose renal histology could be studied. Peritoneal dialysis was the main element of treatment: 43% of children who underwent dialysis improved, compared with only 25% of those who did not undergo dialysis. The haemolytic-uraemic syndrome precipitated by bacillary dystentery is therefore the most important cause of ARF in children aged under 5 years in Tamil Nadu and the adjoining area of Andhra Pradesh.     

Molecular dissection of the beta subunit of F1-ATPase into peptide fragments.

Partial digestion of the native beta subunit of F1-ATPase from the thermophilic Bacillus strain PS3 by three different proteases produced a limited number of peptide fragments. In most cases, the peptides remained associated, and the gross structure of the beta subunit was not destroyed. Furthermore, most peptides were able to reassociate into the form of the beta subunit after denaturating urea treatment. Therefore, the cleaved sites are most likely located in water-exposed loop regions in the tertiary structure of the protein. Almost all peptides were analyzed, and 17 cleaved sites were determined. From the analysis of the distribution of cleaved sites and deletions or insertions in the multiple amino acid sequence alignment of proteins homologous to the beta subunit, locations of five loops and four candidate loops in the beta subunit are suggested. There are two large loops in the central region of the beta subunit sequence, and dicyclohexylcarbodiimide-reactive Glu190 is located in one of them. Tyr341, involved in putative catalytic ATP binding, is also found in one of the loops. Then, taking cleaved sites as a reference, two kinds of expression plasmids, each of which carried genes of two complementary peptide fragments, 1-193 and 198-473 or 1-284 and 285-473, were constructed and expressed in Escherichia coli. For each plasmid, two peptides were coexpressed, associated into a stable beta subunit form in E. coli cells, and purified without dissociation. When these beta subunits were denatured by urea and applied to polyacrylamide gel without denaturant, a protein band with the same mobility as that of the beta subunit appeared, indicating that reassociation of peptide fragments into the form of the beta subunit occurred upon removal of urea. These beta subunits retained the ability to reconstitute the alpha 3 beta 3 gamma complexes even though the efficiency of reconstitution and the recovered ATPase activities were decreased. These complexes were stable at high or low temperature, and ATPase activities were sensitive to inhibition by N3-.     

Inhibition of Maitotoxin-Induced Ca2+ Influx in Rat Glioma C6 Cells by Brevetoxins and Synthetic Fragments of Maitotoxin

Abstract: ⁴⁵Ca²⁺ influx in rat glioma C6 cells induced by 0.3 n M maitotoxin (MTX) was markedly inhibited by brevetoxin A (PbTx1) and brevetoxin B (PbTx2), with EC₅₀ values of 16 and 13 µ M , respectively. This inhibition was observed immediately after addition of MTX when monitored with fura-2, which suggests that PbTx2 directly blocks the action of MTX. This blockade by PbTx2 was not affected by tetrodotoxin, which excludes the involvement of voltage-sensitive sodium channels. The depolarizing effects of these brevetoxins were also not a likely cause of this inhibition, because melittin, a channel-forming peptide, did not significantly block MTX-induced ⁴⁵Ca²⁺ influx. Instead, this inhibition was thought to be mediated by blockade of an MTX-binding site by the brevetoxins, based on the fact that these toxins, particularly PbTx2, closely mimic the partial structure of MTX. Synthetic fragments of MTX corresponding to the hydrophilic EF-GH rings (200 µ M ) and LM-NO rings (500 µ M ) of MTX significantly reduced MTX-elicited Ca²⁺ influx. The observation that the effects of MTX were inhibited by structural mimics of both its hydrophobic and hydrophilic portions implies that both portions of the MTX molecule recognize its target.     

Substituent effects on the PMR signals of methyl groups in methyl steroids

Proton magnetic resonance (PMR) signals were assigned to each of the methyl groups for a variety of C-3 oxygenated 4-monomethyl, 6-monomethyl, 4, 4-dimethyl, and 2, 2-dimethyl steroids related to 5α-cholestane series. The effects of various substituents (i.e. methyls at C-4, C-6, or C-2, functional groups at C-3, and unsaturated bonds) on the methyl proton resonances have been derived from the assignment. The application of such data to structural and stereo-chemical problems has also been discussed.     

The role of transposable element clusters in genome evolution and loss of synteny in the rice blast fungus Magnaporthe oryzae

Background  

Transposable elements are abundant in the genomes of many filamentous fungi, and have been implicated as major contributors to genome rearrangements and as sources of genetic variation. Analyses of fungal genomes have also revealed that transposable elements are largely confined to distinct clusters within the genome. Their impact on fungal genome evolution is not well understood. Using the recently available genome sequence of the plant pathogenic fungus Magnaporthe oryzae, combined with additional bacterial artificial chromosome clone sequences, we performed a detailed analysis of the distribution of transposable elements, syntenic blocks, and other features of chromosome 7.     

Patch-Leaving Decision of the Predatory Mite Amblyseius womersleyi (Acari: Phytoseiidae) Based on Multiple Signals from Both Inside and Outside a Prey Patch

We compared the emigration rates of Amblyseius womersleyi from prey patches (leaf disks) of different conditions in airflow containing either infested plant volatiles (volatiles airflow) or uninfested plant volatiles (control airflow). Both airflow and prey patch conditions significantly affected the emigration rates. Emigration rates from patches carrying prey products (feces, exuviae, webs, etc.) and prey eggs were significantly lower in control airflow than in volatiles airflow. Under other patch conditions, the rates were lower in control airflow than in volatiles airflow, although the difference was not significant. In both airflows, the lowest emigration rates were observed when predators were in a heavily infested patch. Patches carrying prey products and prey eggs resulted in lower emigration rates than patches carrying eggs alone and patches emitting prey-infested plant volatiles but carrying no prey. Thus, A. womersleyi appears to decide the timing of emigration based on two criteria: prey products in the patch and prey-infested plant volatiles from outside.