Protein Dynamics in Complex DNA Lesions

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Per os transmission of iridescent virus of Heliothis zea (Lepidoptera: Noctuidae)

In per os transmission of iridescent virus (IV), the first signs of infection are small iridescent patches in the prolegs, clypeus, labrum, and intersegmental membranes. Per os exposure of neonatal larvae to IV produced 18–40% infection. Per os exposure of 5- and 9-day-old larvae produced 11–47% and 10–30% infection, respectively. A few larvae with a patent infection pupated; however, none reached the adult stage. Infected larvae remained in the larval stage up to 89 days, compared to 12–21 days for normal larvae. Transovum or transovarian transmission was not detected. Examination of fat body, silk glands, and muscles of infected larvae by electron microscopy confirmed the presence of numerous intracytoplasmic virus particles. The mean particle diameter of hexagonal profiles within viral paracrystals was 118±3.5 nm.     

Tubular structures associated with intracytoplasmic spirochetes

When tissues of spirochete-infected brine shrimp (Artemia salina) were examined by electron microscopy, tubular structures were often seen closely associated with intracytoplasmic spirochete profiles. These tubules were never observed in association with spirochetes found in the haemocoeal or other extracellular spaces. Since the trilaminar layer comprising the wall of the tubules was identical in ultrastructure to the outer envelope of the spirochete, these structures were interpreted to be finger-like extensions of the spirochete outer envelope.     

Insulin regulates protein metabolism in mouse blastocysts

Mouse blastocysts, in vitro, endocytosed 100 μg/ml ¹²⁵I-labelled bovine serum albumin (BSA) at a rate equivalent to 192 ± 27 μl/hr/mg embryonic protein over the first 20 min. Insulin stimulated this initial uptake by 30% (P < 0.05). After this time, accumulation of ¹²⁵I-labelled BSA began to plateau as the endocytosed ¹²⁵I-labelled BSA was catabolized and ¹²⁵I was released from the cells. Insulin caused an ≈?72% (P < 0.05) increase in the amount of uncatabolized ¹²⁵I-labelled BSA remaining in insulin-treated blastocysts after 2 hr as compared to control blastocysts. Insulin partially inhibited catabolism of endocytosed ¹²⁵I-labelled BSA during the first 2 hr following transfer to nonradioactive medium. After this time, degradation ceased in both control and insulin-treated blastocysts, leaving a small, uncatabolized protein pool remaining in the embryos; however, as a result of insulin's inhibitory effects on the initial catabolic rate, the uncatabolized protein pool was 30% (P < 0.05) larger in insulin-treated blastocysts after the 4 hr chase. Insulin inhibited endogenous protein degradation in blastocysts by 37% (P < 0.05). Combined with previous studies showing a 90% increase in endogenous protein synthesis in blastocysts following short-term stimulation with insulin (Harvey and Kaye, 1988), these results suggest that insulin acts to increase the endogenous protein-reserves in the embryo. Dose-response studies indicated an EC₅₀ of 0.5 pM for insulin's stimulation of ¹²⁵I-labelled BSA accumulation, consistent with action via its own receptor. Insulin-like growth factor-1 (IGF-1) also stimulated protein accumulation at concentrations similar to those observed with insulin, suggesting that IGF-1 may act via its own receptor rather than the insulin receptor to exert its effects on endocytosis. © 1993 Wiley-Liss, Inc.     

The first independent Ukrainian census in Crimea: Myths,miscoding, and missed opportunities

State-defined identity categories can have a profound impact on individuals’ conception of themselves. Like birth certificates and migration documents, the census is a crucial instrument in producing and maintaining ethnic and racial identities. Recent research suggests that censuses measure preferences, rather than objective data, and can profitably be studied along the lines of political campaigns. This article advances the idea that the next question is whose preference is being recorded. Ethnographic research on the micropolitics of census-taking in Crimea, Ukraine suggest the dynamics between census-takers and ethnic constituencies, as well as instructions from census officials with various ethnic loyalties, have a crucial role to play.     

Caribbean golden orbweaving spiders maintain gene flow with North America

The Caribbean archipelago offers one of the best natural arenas for testing biogeographic hypotheses. The intermediate dispersal model of biogeography (IDM) predicts variation in species richness among lineages on islands to relate to their dispersal potential. To test this model, one would need background knowledge of dispersal potential of lineages and their biogeographic patterns, which has been problematic as evidenced by our prior work on the Caribbean tetragnathid spiders. In order to investigate the biogeographic imprint of an excellent disperser, we study Trichonephila in the Americas. Trichonephila is a nephilid genus that contains globally distributed species known to overcome long, overwater distances. The results of our phylogenetic and population genetic analyses on T. clavipes suggest that populations over the Caribbean and North America maintain a lively gene flow. However, the single species status of T. clavipes over the entire New World is challenged by our species delimitation analyses. Combined with prior evidence from spider genera of different dispersal ability, these patterns coming from an excellent disperser (Trichonephila) that is species-poor and of a relatively homogenous genetic structure, support the IDM predictions.     

Ebolavirus and Marburgvirus: insight the Filoviridae family

Ebolavirus and Marburgvirus (belonging to the Filoviridae family) emerged four decades ago and cause epidemics of haemorrhagic fever with high case-fatality rates. The genome of filoviruses encodes seven proteins. No significant homology is observed between filovirus proteins and any known macromolecule. Moreover, Marburgvirus and Ebolavirus show significant differences in protein homology. The natural maintenance cycle of filoviruses is unknown, the natural reservoir, the mode of transmission, the epidemic disease generation, and temporal dynamics are unclear. Lastly, Ebolavirus and Marburgvirus are considered as potential biological weapons. Vaccine appears the unique therapeutic frontier. Here, molecular and clinical aspects of filoviral haemorrhagic fevers are summarized.     

Efficient and inexpensive transient expression of multispecific multivalent antibodies in Expi293 cells

Background

Immunotherapy is a very fast expanding field within drug discovery and, hence, rapid and inexpensive expression of antibodies would be extremely valuable. Antibodies are, however, difficult to express. Multifunctional antibodies with additional binding domains further complicate the expression. Only few protocols describe the production of tetravalent bispecific antibodies and all with limited expression levels.?

Methods

Here, we describe a protocol that can produce functional tetravalent, bispecific antibodies at around 22 mg protein/l to a low cost. The expression system is based on the Expi293 cells, which have been adapted to grow in denser cultures than HEK293 cells and gives higher expression yields. The new protocol transfects the E?xpi293 cells with PEI (which has a negligible cost).

Results

The protocol has been used to generate multiple variants of tetra- and hexavalent bispecific antibodies with yields of around 22 mg protein/l within 10 days. All materials are commercially available and the implementation of the protocol is inexpensive and straightforward. The bispecific antibodies generated in our lab were capable of binding to all antigens with similar affinity as the original antibody. Two of the bispecific antibodies have also been used in transgenic mice as positron emission tomography (PET) ligands to successfully detect amyloid-beta (Aβ) aggregates in vivo.

Conclusions

This protocol is the first describing transfection of the human Expi293 cells with PEI. It can be used to generate functional multi-specific antibodies in high amounts. The use of biological drugs, and in particular multispecific antibodies, is rapidly increasing, hence improved protocols such as the one presented here are highly valuable.