The mink gene for the λ light immunoglobulin chain: Characterization of cDNA and chromosomal localization

A cDNA library from mink spleen was constructed by use of the phage gt11. The library was screened using polyvalent serum raised against the mink immunoglobulin chain. As a result, several clones expressing mink immunoglobulin light chains were identified. Sequencing of one of the clones with an 803 bp insert was performed. The insert comprised nearly the entire coding region for the mature light immunoglobulin gene with the exception of the leader polypeptide and several amino acids of the RF1 region of the V segment. Compared with the rabbit, mouse and human light immunoglobulin genes, the homology of the cloned sequence was found to be highest relative to the rabbit gene. With the cloned mink cDNA containing the C-region only as a probe, the DNAs from mink-Chinese hamster hybrid clones were studied. The results of segregation analysis of this mink cDNA sequence and mink chromosomes in the mink-Chinese hamster clone panel allowed us to assign the gene for the light immunoglobulin constant polypeptide (IGLC) to mink Chromosome (Chr) 4.     

Determinants of protein function revealed by combinatorial entropy optimization

We use a new algorithm (combinatorial entropy optimization [CEO]) to identify specificity residues and functional subfamilies in sets of proteins related by evolution. Specificity residues are conserved within a subfamily but differ between subfamilies, and they typically encode functional diversity. We obtain good agreement between predicted specificity residues and experimentally known functional residues in protein interfaces. Such predicted functional determinants are useful for interpreting the functional consequences of mutations in natural evolution and disease.     

Migration promotes mutator alleles in subdivided populations

Mutator alleles that elevate the genomic mutation rate may invade nonrecombining populations by hitchhiking with beneficial mutations. Mutators have been repeatedly observed to take over adapting laboratory populations and have been found at high frequencies in both microbial pathogen and cancer populations in nature. Recently, we have shown that mutators are only favored by selection in sufficiently large populations and transition to being disfavored as population size decreases. This population size‐dependent sign inversion in selective effect suggests that population structure may also be an important determinant of mutation rate evolution. Although large populations may favor mutators, subdividing such populations into sufficiently small subpopulations (demes) might effectively inhibit them. On the other hand, migration between small demes that otherwise inhibit hitchhiking may promote mutator fixation in the whole metapopulation. Here, we use stochastic, agent‐based simulations and evolution experiments with the yeast Saccharomyces cerevisiae to show that mutators can, indeed, be favored by selection in subdivided metapopulations composed of small demes connected by sufficient migration. In fact, we show that population structure plays a previously unsuspected role in promoting mutator success in subdivided metapopulations when migration is rare.     

Commercial ampholytes used for isoelectric focusing may interfere with bioactivity based purification of antimicrobial peptides

BioRad's Rotofor system has been frequently used for the purification of proteins and smaller peptides such as bacteriocins. In this study, we report that some commercially available ampholytes used with the Rotofor isoelectric focusing system possess antimicrobial activity, which may interfere with the purification of bacteriocins and bacteriocin-like substances.     

Study of the mechanism of Ras-dva small GTPase intracellular localization

An analysis of amino acid sequences of small GTPases of the Ras-dva family allowed us to determine the C-terminal prenylation motif, which could be responsible for the membrane localization of these proteins. We demonstrated using in vivo EGFP tracing that the Ras-dva small GTPases from Xenopus laevis embryo cells and NIH-3T3 fibroblasts are localized on both plasma membranes and endomembranes (the endoplasmic reticulum, the Golgi apparatus, and vesicles). At the same time, the replacement of the Cys residue, the SH group of which must be theoretically farnesylated, in the C-terminal prenylation motif of the Ras-dva small GTPase by the Ser residue prevented the membrane localization of the protein. These results indicate that the C-terminal prenylation site is critical for the membrane localization of small Ras-dva GTPases.     

Role of cooperative cell movements and mechano-geometric constrains in patterning of axial rudiments in Xenopus laevis embryos

The role of cooperative cell movements has been explored in establishment of regular segregation of the marginal zone of Xenopus laevis embryos into the main axial rudiments: notochord, somites and neural tissue. For this purpose, the following operations were performed at the late blastula-early gastrula stages: (1) isolation of marginal zones, (2) addition of the ventral zone fragments to the marginal zones, (3) dissection of isolated marginal zones along either ventral (a) or dorsal (b) midlines, (4) immediate retransplantation of excised fragments of the suprablastoporal area to the same places without rotation or after 90 degrees rotation, (5) pi-shaped separation of the suprablastoporal area either anteriorly or posteriorly. In experiments 1, 4, and 5, lateromedial convergent cell movements and differentiation of the axial rudiments were suppressed. In experiments 4 and 5, cell movements were reoriented ventrally, the entire embryo architecture was extensively reconstructed, and the axial rudiments were relocated to the blastopore lateral lips. In experiment 3, convergent cell movements were restored and oriented either towards the presumptive embryo midline (a), or in the perpendicular direction (b). In both cases, well developed axial rudiments elongated perpendicularly to cell convergences were formed. If the areas of axial rudiment formation were curved, mesodermal somites and neural tissue were always located on the convex (stretched) and concave (compressed) sides, respectively. We conclude that no stable prepatterning of the marginal zone takes place until at least the midgastrula stage. This prepatterning requires cooperative cell movements and associated mechano-geometric constrains.     

Nonlinear‐optical stain‐free stereoimaging of astrocytes and gliovascular interfaces

Methods of nonlinear optics provide a vast arsenal of tools for label‐free brain imaging, offering a unique combination of chemical specificity, the ability to detect fine morphological features, and an unprecedentedly high, subdiffraction spatial resolution. While these techniques provide a rapidly growing platform for the microscopy of neurons and fine intraneural structures, optical imaging of astroglia still largely relies on filament‐protein‐antibody staining, subject to limitations and difficulties especially severe in live‐brain studies. Once viewed as an ancillary, inert brain scaffold, astroglia are being promoted, as a part of an ongoing paradigm shift in neurosciences, into the role of a key active agent of intercellular communication and information processing, playing a significant role in brain functioning under normal and pathological conditions. Here, we show that methods of nonlinear optics provide a unique resource to address long‐standing challenges in label‐free astroglia imaging. We demonstrate that, with a suitable beam‐focusing geometry and careful driver‐pulse compression, microscopy of second‐harmonic generation (SHG) can enable a high‐resolution label‐free imaging of fibrillar structures of astrocytes, most notably astrocyte processes and their endfeet. SHG microscopy of astrocytes is integrated in our approach with nonlinear‐optical imaging of red blood cells based on third‐harmonic generation (THG) enhanced by a three‐photon resonance with the Soret band of hemoglobin. With astroglia and red blood cells providing two physically distinct imaging contrasts in SHG and THG channels, a parallel detection of the second and third harmonics enables a high‐contrast, high‐resolution, stain‐free stereoimaging of gliovascular interfaces in the central nervous system. Transverse scans of the second and third harmonics are shown to resolve an ultrafine texture of blood‐vessel walls and astrocyte‐process endfeet on gliovascular interfaces with a spatial resolution within 1 μm at focusing depths up to 20 μm inside a brain.     

3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures

DNA probes with conjugated minor groove binder (MGB) groups form extremely stable duplexes with single-stranded DNA targets, allowing shorter probes to be used for hybridization based assays. In this paper, sequence specificity of 3′-MGB probes was explored. In comparison with unmodified DNA, MGB probes had higher melting temperature (T_m) and increased specificity, especially when a mismatch was in the MGB region of the duplex. To exploit these properties, fluorogenic MGB probes were prepared and investigated in the 5′-nuclease PCR assay (real-time PCR assay, TaqMan assay). A 12mer MGB probe had the same T_m (65°C) as a no-MGB 27mer probe. The fluorogenic MGB probes were more specific for single base mismatches and fluorescence quenching was more efficient, giving increased sensitivity. A/T rich duplexes were stabilized more than G/C rich duplexes, thereby leveling probe T_m and simplifying design. In summary, MGB probes were more sequence specific than standard DNA probes, especially for single base mismatches at elevated hybridization temperatures.     

Innate mechanisms of viral recognition

Viruses are obligate parasites which can infect cells of all living organisms. Multiple antiviral defense mechanisms appeared early in the evolution of the immune system. Higher vertebrates possess the most complex antiviral immunity based on both innate and adoptive immune responses. However, a majority of living organisms, including plants and invertebrates, rely exclusively on innate immune mechanisms for protection against viral infections. There are some striking similarities in several components of innate immune recognition in mammals, plants, and insects suggesting that these signaling cascades are highly conserved in the evolution of the immune system. This review summarizes recent advances in the field of innate immune recognition of viruses, with a focus on pattern-recognition receptors.