Limited proteolysis patterns of the B chain of insulin.

The oxidized B chain of insulin was used as a simple model for further consideration of limited proteolysis with low substrate:enzyme ratios. With low B chain:trypsin ratios, the ordinarily slower cleavage rate of the -Lys₂₉-Ala₃₀ bond essentially equaled the cleavage saturation rate of the -Arg₂₂-Gly₂₃ bond. This led to the disappearance of octapeptide which ordinarily forms most rapidly. Heptapeptide and alanine, formed mainly by cleavage of the octapeptide, decreased somewhat at high enzyme relative levels. Trypsin added to B chain formed a single chromatographic peak.     

Identification of significant regions of transcription factor DP-1 (TFDP-1) involved in stability/instability of the protein

We previously reported the identification of DP-1 isoforms (α and β), which are structurally C-terminus-deleted ones, and revealed the low-level expression of these isoforms. It is known that wild-type DP-1 is degraded by the ubiquitin-proteasome system, but few details are known about the domains concerned with the protein stability/instability for the proteolysis of these DP-1 isoforms. Here we identified the domains responsible for the stability/instability of DP-1. Especially, the DP-1 “Stabilon” domain was a C-terminal acidic motif and was quite important for DP-1 stability. Moreover, we propose that this DP-1 Stabilon may be useful for the stability of other nuclear proteins when fused to them.     

Generation of a purely clonal defective interfering influenza virus

Defective interfering (DI) influenza viruses carry a large deletion in a gene segment that interferes with the replication of infectious virus; thus, such viruses have potential for antiviral therapy. However, because DI viruses cannot replicate autonomously without the aid of an infectious helper virus, clonal DI virus stocks that are not contaminated with helper virus have not yet been generated. To overcome this problem, we used reverse genetics to generate a clonal DI virus with a PB2 DI gene, amplified the clonal DI virus using a cell line stably expressing the PB2 protein, and confirmed its ability to interfere with infectious virus replication in vitro. Thus, our approach is suitable for obtaining purely clonal DI viruses, will contribute to the understanding of DI virus interference mechanisms and can be used to develop DI virus‐based antivirals.     

Zearalenone induces chromosome aberrations in mouse bone marrow: preventive effect of 17β-estradiol, progesterone and Vitamin E

The cytogenetic effect of zearalenone (ZEN), a non-steroidal estrogenic mycotoxin, was evaluated in vivo, in mouse bone marrow cells, by assessing the percentage of cells bearing different chromosome aberrations. The studies included different conditions for animal treatment, as follows: (1) single intraperitoneal (ip) injection, (2) repeated ip injections, (3) pre-treatment for 24 h with Vitamin E (Vit E), and (4) pre-treatment for 4 h with 17β-estradiol (17β-Est) or progesterone (Prog). ZEN induced different types of chromosome aberrations, which was concentration-dependent (2–20 mg/kg bw). These doses corresponded to 0.4–4% of the LD₅₀ in the mouse. Interestingly, when the dose of ZEN (40 mg/kg) was fractionated into four equivalent doses (4 × 10 mg/kg bw), into three doses (15 + 10 + 15 mg/kg bw), or into two equivalent doses (2 × 20 mg/kg bw), given every 24 h, the percentage of chromosome aberrations increased significantly. This finding suggests that ZEN proceeds by reversible binding on receptors that could become saturated, and that it damages the chromosomes in a ‘hit and go’ manner. Furthermore, pre-treatment of animals with 17β-estradiol or progesterone significantly decreased the percentage of chromosome aberrations, suggesting that (i) these hormones bind to the same cytoplasmic receptors transported into the nucleus to elicit DNA damage, (ii) they may play a role in preventing chromosome aberrations induced by ZEN. Similarly, Vit E prevented these chromosome aberrations indicating that Vit E, previously reported to prevent most of the toxic effects induced by ZEN, may also bind to the same receptors.     

Site-Specific Structural Variations Accompanying Tubular Assembly of the HIV-1 Capsid Protein

The 231-residue capsid (CA) protein of human immunodeficiency virus type 1 (HIV-1) spontaneously self-assembles into tubes with a hexagonal lattice that is believed to mimic the surface lattice of conical capsid cores within intact virions. We report the results of solid-state nuclear magnetic resonance (NMR) measurements on HIV-1 CA tubes that provide new information regarding changes in molecular structure that accompany CA self-assembly, local dynamics within CA tubes, and possible mechanisms for the generation of lattice curvature. This information is contained in site-specific assignments of signals in two- and three-dimensional solid-state NMR spectra, conformation-dependent ¹⁵N and ¹³C NMR chemical shifts, detection of highly dynamic residues under solution NMR conditions, measurements of local variations in transverse spin relaxation rates of amide ¹H nuclei, and quantitative measurements of site-specific ¹⁵N–¹⁵N dipole–dipole couplings. Our data show that most of the CA sequence is conformationally ordered and relatively rigid in tubular assemblies and that structures of the N-terminal domain (NTD) and the C-terminal domain (CTD) observed in solution are largely retained. However, specific segments, including the N-terminal β-hairpin, the cyclophilin A binding loop, the inter-domain linker, segments involved in intermolecular NTD–CTD interactions, and the C-terminal tail, have substantial static or dynamical disorder in tubular assemblies. Other segments, including the 3₁₀-helical segment in CTD, undergo clear conformational changes. Structural variations associated with curvature of the CA lattice appear to be localized in the inter-domain linker and intermolecular NTD–CTD interface, while structural variations within NTD hexamers, around local 3-fold symmetry axes, and in CTD–CTD dimerization interfaces are less significant.     

Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.     

Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280–320-kDa oligomeric structure consisting of ∼6–7 subunits.     

Molecular Identification of Aeromonas and Coliform Bacteria Isolated on m Endo Media from Lake Erie Waters

Summary Bacteria from recreational waters collected from two Lake Erie beaches in Dunkirk, New York were plated onto m Endo LES media. The 16S rRNA gene was then amplified from coliform and non-coliform bacteria using the polymerase chain reaction. The PCR products were characterized by restriction fragment length polymorphism (RFLP) analysis. A total of 8 RFLP groups were identified from the analysis of 920 samples and selected PCR products from each group were sequenced. The DNA sequence analysis indicated that more than half of the bacteria identified as coliforms on the m Endo plates belonged to the genus Aeromonas from the family Aeromonadaceae. Most of the remaining coliforms were from the Enterobacteriaceae. The data indicate that m Endo agar plates allow the growth of non-coliform bacteria, especially Aeromonas species.