Noncovalent Immobilization of Streptavidin on In Vitro- and In Vivo-Biotinylated Bacterial Magnetic Particles

Biotinylated magnetic nanoparticles were constructed by displaying biotin acceptor peptide (BAP) or biotin carboxyl carrier protein (BCCP) on the surface of bacterial magnetic particles (BacMPs) synthesized by Magnetospirillum magneticum AMB-1. BAP-displaying BacMPs (BAP-BacMPs) were extracted from bacterial cells and incubated with biotin and Escherichia coli biotin ligase. Then the in vitro biotinylation of BAP-BacMPs was confirmed using alkaline phosphatase-labeled antibiotin antibody. In contrast, BacMPs displaying the intact 149 residues of AMB-1 BCCP (BCCP-BacMPs) and displaying the COOH-terminal 78 residues of BCCP (BCCP78-BacMPs) were biotinylated in AMB-1 cells. The in vivo biotinylation of BCCP-BacMPs and BCCP78-BacMPs was thought to be performed by endogenous AMB-1 biotin ligase. Streptavidin was introduced onto biotinylated BacMPs by simple mixing. In an analysis using tetramethyl rhodamine isocyanate-labeled streptavidin, approximately 15 streptavidin molecules were shown to be immobilized on a single BCCP-BacMP. Furthermore, gold nanoparticle-BacMP composites were constructed via the biotin-streptavidin interaction. The conjugation system developed in this work provides a simple, low-cost method for producing biotin- or streptavidin-labeled magnetic nanoparticles. Various functional materials can be site selectively immobilized on these specially designed BacMPs. By combining the site-selective biotinylation technology and the protein display technology, more innovative and attractive magnetic nanomaterials can be constructed.     

Simian Virus 40 Vp1 DNA-Binding Domain Is Functionally Separable from the Overlapping Nuclear Localization Signal and Is Required for Effective Virion Formation and Full Viability

A DNA-binding domain (DBD) was identified on simian virus 40 (SV40) major capsid protein Vp1, and the domain's function in the SV40 life cycle was examined. The DBD was mapped by assaying various recombinant Vp1 proteins for DNA binding in vitro. The carboxy-terminal 58-residue truncated Vp1DeltaC58 pentamer bound DNA with a K(d) of 1.8 x 10(-9) M in terms of the protein pentamer, while full-length Vp1 and carboxy-terminal-17-truncated Vp1DeltaC17 had comparable apparent K(d)s of 5.3 x 10(-9) to 7.3 x 10(-9) M in terms of the protein monomers. Previously identified on Vp1 was a nuclear localization signal (NLS) consisting of two N-terminal basic clusters, NLS1 (4-KRK-6) and NLS2 (15-KKPK-18). Vp1DeltaC58 pentamers harboring multiple-point mutations in NLS1 (NLSm1), NLS2 (NLSm2), or both basic clusters (NLSm1. 2) had progressively decreased DNA-binding activity, down to 0.7% of the Vp1DeltaC58 level for NLSm1. 2 Vp1. These data, along with those of N-terminally truncated proteins, placed the DBD in overlap with the bipartite NLS. The role of the Vp1 DBD during infection was investigated by taking advantage of NLS phenotypic complementation (N. Ishii, A. Nakanishi, M. Yamada, M. H. Macalalad, and H. Kasamatsu, J. Virol. 68:8209-8216, 1994), in which an NLS-defective Vp1 could localize to the nucleus in the presence of wild-type minor capsid proteins Vp2 and Vp3. This approach made it possible to dissect the role of the bifunctional Vp1 NLS-DBD in virion assembly in the nucleus. Mutants of the viable nonoverlapping SV40 (NO-SV40) DNA NLSm1, NLSm2, and NLSm1. 2 replicated normally following transfection into host cells and produced capsid proteins at normal levels. All mutant Vp1s were able to interact with Vp3 in vitro. The mutants NLSm1 and NLSm1. 2 were nonviable, and the mutant Vp1s unexpectedly failed to localize to the nucleus though Vp2 and Vp3 did, suggesting that the mutated NLS1 acted as a dominant signal for the cytoplasmic localization of Vp1. Mutant NLSm2, for which the mutant Vp1's nuclear localization defect was complemented by Vp2 and Vp3, displayed a 5,000-fold reduced viability. Analysis of NLSm2 DNA-transfected cell lysate revealed a 10-fold reduction in the level of DNase I-protected viral DNA, and yet virion-like particles were found among the DNase I-resistant material. Collective results support a role for Vp1 NLS2-DBD2 in the assembly of virion particles. The results also suggest that this determinant can function in the infection of new cells.     

A River Metapopulation Structure of a Japanese Freshwater Goby, Odontobutis Obscura, Deduced from Allozyme Genetic Indices

Gene products of 18 allozyme loci from 1268 individuals of a Japanese freshwater goby called donko, Odontobutis obscura (Odontobutidae; Gobioidei), from 33 localities in the Koya River, Yamaguchi Prefecture, Japan, were investigated to determine the extent of genetic divergence and gene flow within a river metapopulation. Genetic indices including GST(mean FST 0.182), FIT(mean 0.192) and D(mean 0.015) indicated a considerable divergence of local populations in the river. The genetic distance (D) and channel distance between pairs of populations did not show a good correlation, and geographical neighbors were not always genetic neighbors. Therefore, the genetic divergence of populations is attributable to independent genetic drift with restricted gene flow among populations. The agricultural dams and weirs constructed across the river must be responsible for the restricted gene flow. The metapopulation structure of O. obscura in the Koya River may be barely sustained by one-way gene flow only from the upper to the lower populations. An occasional artificial transplantation of some individuals from the lower to the upper populations may be one alternative to maintain a river metapopulation structure safely.     

Localization of swine PRE-1 homologues in 13 loci of Phacochoerus aethiopicus and Tayassu tajacu genomes, and their sequence divergence

In our previous study, PRE-1 (a swine short interspersed nuclear element, SINE) was found to be present in the genomes of animal species related to swine ( Sus scrofa ) i.e. warthog ( Phacochoerus aethiopicus ) and collared peccary ( Tayassu tajacu ) at almost the same frequency as in Sus scrofa . In the present study, we investigated whether PRE-1 was present in hippopotamus ( Hippopotamus amphibius ), which is in the same order but in a different family to Sus scrofa . Hippopotamus amphibius was found to contain no PRE-1. Then, in order to study the localization of PRE-1 sequences at locus level and the sequence divergence of the PRE-1 of individual loci among Sus scrofa , Phacochoerus aethiopicus and Tayassu tajacu , primer sets, which can amplify PRE-1 sequences at 13 loci of swine genome with the polymerase chain reaction, were prepared to identify any corresponding sequences in Phacochoerus aethiopicus and Tayassu tajacu . Twelve and nine of the 13 primer sets identified fragments in Phacochoerus aethiopicus and Tayassu tajacu respectively. Ten of the 12 Phacochoerus aethiopicus fragments and two of the nine Tayassu tajacu fragments contained PRE-1 sequences. Based on the divergence between the corresponding PRE-1 sequences at individual loci and on the mutation rate of the pseudogenes (r=4·6×10^–9), Phacochoerus aethiopicus and Tayassu tajacu are currently calculated to have been separated from Sus scrofa later than 1·4million years before present (MYBP) and 16·8 MYBP, respectively.     

Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride

Enthalpy and heat capacity changes for the deprotonation of 18 buffers were calorimetrically determined in 0.1 M potassium chloride at temperatures ranging from 5 to 45°C. The values of the dissociation constant were also determined by means of potentiometric titration. The enthalpy changes for the deprotonation of buffers, except for the phosphate and glycerol 2-phosphate buffers, were found to be characterized by a linear function of temperature. The enthalpy changes for the second dissociation of phosphate and glycerol 2-phosphate where divalent anion is formed on dissociation were fitted with the second order function of temperature rather than the first order. Temperature dependence of buffer pH calculated by using the enthalpy and heat capacity changes obtained was in good agreement with the temperature variation of the pH values actually measured in the temperature range between 0 and 50°C for all the buffers studied. On the basis of the results obtained, a numeric table showing the temperature dependence of pK values for the 18 buffers is presented. Proteins 33:159–166, 1998. © 1998 Wiley-Liss, Inc.     

Removal of gelatin from live vaccines and DTaP—an ultimate solution for vaccine-related gelatin allergy

From the early 1990s infants started to receive acellular pertussis vaccine combined with diphtheria and tetanus toxoids (DTaP) before live vaccines such as measles, rubella, and mumps vaccines, which contained gelatin as a stabilizer. Then, an increasing number of cases of anaphylactic/allergic reactions to those live vaccines were reported. Almost all these cases had a previous history of receiving three or four doses of DTaP containing gelatin.Anaphylactic/allergic reactions to live measles vaccine were analyzed using information obtained from the Reporting System, a retrospective study, as well as from the Monitoring System, a prospective study. Dramatic decreases in anaphylactic/allergic reactions to live measles vaccines were observed immediately after each manufacturer marketed gelatin-free or gelatin (hypo-allergic)-containing live measles vaccine, and since the end of 1998 reports on anaphylactic/allergic reactions to live measles vaccine have almost ceased.     

Immature Pacific bluefin tuna, <Emphasis Type="Italic">Thunnus orientalis</Emphasis>, utilizes cold waters in the Subarctic Frontal Zone for trans-Pacific migration

The habitat and movements of a Pacific bluefin tuna were investigated by reanalyzing archival tag data with sea surface temperature data. During its trans-Pacific migration to the eastern Pacific, the fish took a direct path and primarily utilized waters, in the Subarctic Frontal Zone (SFZ). Mean ambient temperature during the trans-Pacific migration was 14.5 ± 2.9 (°C ± SD), which is significantly colder than the waters typically inhabited by bluefin tuna in their primary feeding grounds in the western and eastern Pacific (17.6 ± 2.1). The fish moved rapidly through the colder water, and the heat produced during swimming and the thermoconservation ability of bluefin tuna likely enabled it to migrate through the cold waters of the SFZ.