In vitro recombination of non-homologous genes can result in gene fusions that confer a switching phenotype to cells

Regulation of protein activity is central to the complexity of life. The ability to regulate protein activity through exogenously added molecules has biotechnological/biomedical applications and offers tools for basic science. Such regulation can be achieved by establishing a means to modulate the specific activity of the protein (i.e. allostery). An alternative strategy for intracellular regulation of protein activity is to control the amount of protein through effects on its production, accumulation, and degradation. We have previously demonstrated that the non-homologous recombination of the genes encoding maltose binding protein (MBP) and TEM1 β-lactamase (BLA) can result in fusion proteins in which β-lactamase enzyme activity is allosterically regulated by maltose. Here, through use of a two-tiered genetic selection scheme, we demonstrate that such recombination can result in genes that confer maltose-dependent resistance to β-lactam even though they do not encode allosteric enzymes. These 'phenotypic switch' genes encode fusion proteins whose accumulation is a result of a specific interaction with maltose. Phenotypic switches represent an important class of proteins for basic science and biotechnological applications in vivo.     

An antibody to the lutheran glycoprotein (Lu) recognizing the LU4 blood type variant inhibits cell adhesion to laminin α5

Background

The Lutheran blood group glycoprotein (Lu), an Ig superfamily (IgSF) transmembrane receptor, is also known as basal cell adhesion molecule (B-CAM). Lu/B-CAM is a specific receptor for laminin α5, a major component of basement membranes in various tissues. Previous reports have shown that Lu/B-CAM binding to laminin α5 contributes to sickle cell vaso-occlusion. However, as there are no useful tools such as function-blocking antibodies or drugs, it is unclear how epithelial and sickled red blood cells adhere to laminin α5 via Lu/B-CAM.

Methodology/Principal Findings

In this study, we discovered a function-blocking antibody that inhibits Lu binding to laminin α5 using a unique binding assay on tissue sections. To characterize the function-blocking antibody, we identified the site on Lu/B-CAM recognized by this antibody. The extracellular domain of Lu/B-CAM contains five IgSF domains, D1-D2-D3-D4-D5. The antibody epitope was localized to D2, but not to the D3 domain containing the major part of the laminin α5 binding site. Furthermore, mutagenesis studies showed that Arg¹⁷⁵, the LU4 blood group antigenic site, was crucial for forming the epitope and the antibody bound sufficiently close to sterically hinder the interaction with α5. Cell adhesion assay using the antibody also showed that Lu/B-CAM serves as a secondary receptor for the adhesion of carcinoma cells to laminin α5.

Conclusion/Significance

This function-blocking antibody against Lu/B-CAM should be useful for not only investigating cell adhesion to laminin α5 but also for developing drugs to inhibit sickle cell vaso-occlusion.     

Common variants in a novel gene, FONG on chromosome 2q33.1 confer risk of osteoporosis in Japanese

Osteoporosis is a common disease characterized by low bone mass, decreased bone quality and increased predisposition to fracture. Genetic factors have been implicated in its etiology; however, the specific genes related to susceptibility to osteoporosis are not entirely known. To detect susceptibility genes for osteoporosis, we conducted a genome-wide association study in Japanese using ∼270,000 SNPs in 1,747 subjects (190 cases and 1,557 controls) followed by multiple levels of replication of the association using a total of ∼5,000 subjects (2,092 cases and 3,114 controls). Through these staged association studies followed by resequencing and linkage disequilibrium mapping, we identified a single nucleotide polymorphism (SNP), rs7605378 associated with osteoporosis. (combined P = 1.51×10⁻⁸, odds ratio = 1.25). This SNP is in a previously unknown gene on chromosome 2q33.1, FONG. FONG is predicted to encode a 147 amino-acid protein with a formiminotransferase domain in its N-terminal (FTCD_N domain) and is ubiquitously expressed in various tissues including bone. Our findings would give a new insight into osteoporosis etiology and pathogenesis.     

Drosophila carrying pex3 or pex16 mutations are models of Zellweger syndrome that reflect its symptoms associated with the absence of peroxisomes

The peroxisome biogenesis disorders (PBDs) are currently difficult-to-treat multiple-organ dysfunction disorders that result from the defective biogenesis of peroxisomes. Genes encoding Peroxins, which are required for peroxisome biogenesis or functions, are known causative genes of PBDs. The human peroxin genes PEX3 or PEX16 are required for peroxisomal membrane protein targeting, and their mutations cause Zellweger syndrome, a class of PBDs. Lack of understanding about the pathogenesis of Zellweger syndrome has hindered the development of effective treatments. Here, we developed potential Drosophila models for Zellweger syndrome, in which the Drosophila pex3 or pex16 gene was disrupted. As found in Zellweger syndrome patients, peroxisomes were not observed in the homozygous Drosophila pex3 mutant, which was larval lethal. However, the pex16 homozygote lacking its maternal contribution was viable and still maintained a small number of peroxisome-like granules, even though PEX16 is essential for the biosynthesis of peroxisomes in humans. These results suggest that the requirements for pex3 and pex16 in peroxisome biosynthesis in Drosophila are different, and the role of PEX16 orthologs may have diverged between mammals and Drosophila. The phenotypes of our Zellweger syndrome model flies, such as larval lethality in pex3, and reduced size, shortened longevity, locomotion defects, and abnormal lipid metabolisms in pex16, were reminiscent of symptoms of this disorder, although the Drosophila pex16 mutant does not recapitulate the infant death of Zellweger syndrome. Furthermore, pex16 mutants showed male-specific sterility that resulted from the arrest of spermatocyte maturation. pex16 expressed in somatic cyst cells but not germline cells had an essential role in the maturation of male germline cells, suggesting that peroxisome-dependent signals in somatic cyst cells could contribute to the progression of male germ-cell maturation. These potential Drosophila models for Zellweger syndrome should contribute to our understanding of its pathology.     

A role for fetal hemoglobin and maternal immune IgG in infant resistance to Plasmodium falciparum malaria

Background

In Africa, infant susceptibility to Plasmodium falciparum malaria increases substantially as fetal hemoglobin (HbF) and maternal immune IgG disappear from circulation. During the first few months of life, however, resistance to malaria is evidenced by extremely low parasitemias, the absence of fever, and the almost complete lack of severe disease. This resistance has previously been attributed in part to poor parasite growth in HbF-containing red blood cells (RBCs). A specific role for maternal immune IgG in infant resistance to malaria has been hypothesized but not yet identified.

Methods and Findings

We found that P. falciparum parasites invade and develop normally in fetal (cord blood, CB) RBCs, which contain up to 95% HbF. However, these parasitized CB RBCs are impaired in their binding to human microvascular endothelial cells (MVECs), monocytes, and nonparasitized RBCs – cytoadherence interactions that have been implicated in the development of high parasite densities and the symptoms of malaria. Abnormal display of the parasite''s cytoadherence antigen P. falciparum erythrocyte membrane protein-1 (PfEMP-1) on CB RBCs accounts for these findings and is reminiscent of that on HbC and HbS RBCs. IgG purified from the plasma of immune Malian adults almost completely abolishes the adherence of parasitized CB RBCs to MVECs.

Conclusions

Our data suggest a model of malaria protection in which HbF and maternal IgG act cooperatively to impair the cytoadherence of parasitized RBCs in the first few months of life. In highly malarious areas of Africa, an infant''s contemporaneous expression of HbC or HbS and development of an immune IgG repertoire may effectively reconstitute the waning protective effects of HbF and maternal immune IgG, thereby extending the malaria resistance of infancy into early childhood.     

Brain‐derived neurotrophic factor,corticotropin‐releasing factor,and hypothalamic neuronal histamine interact to regulate feeding behavior

Brain‐derived neurotrophic factor (BDNF), corticotropin‐releasing factor (CRF), and hypothalamic neuronal histamine are anorexigenic substances within the hypothalamus. This study examined the interactions among BDNF, CRF, and histamine during the regulation of feeding behavior in rodents. Food intake was measured after treatment with BDNF, α‐fluoromethyl histidine (FMH; a specific suicide inhibitor of histidine decarboxylase that depletes hypothalamic neuronal histamine), or CRF antagonist. We measured food intake in wild‐type mice and mice with targeted disruption of the histamine H1 receptor (H1KO mice) after central BDNF infusion. Furthermore, we investigated CRF content and histamine turnover in the hypothalamus after BDNF treatment, and conversely, BDNF content in the hypothalamus after histamine treatment. We used immunohistochemical staining for histamine H1 receptors (H1‐R) in BDNF neurons. BDNF‐induced feeding suppression was partially attenuated in rats pre‐treated with FMH or a CRF antagonist, and in H1KO mice. BDNF treatment increased CRF content and histamine turnover in the hypothalamus. Histamine increased BDNF content in the hypothalamus. Immunohistochemical analysis revealed that H1‐Rs were expressed on BDNF neurons in the ventromedial nucleus of the hypothalamus. These results indicate that CRF and hypothalamic neuronal histamine mediate the suppressive effects of BDNF on feeding behavior and body weight.     

Golgi inheritance in the primitive red alga, Cyanidioschyzon merolae

The Golgi body has important roles in modifying, sorting, and transport of proteins and lipids. Eukaryotic cells have evolved in various ways to inherit the Golgi body from mother to daughter cells, which allows the cells to function properly immediately after mitosis. Here we used Cyanidioschyzon merolae, one of the most suitable systems for studies of organelle dynamics, to investigate the inheritance of the Golgi. Two proteins, Sed5 and Got1, were used as Golgi markers. Using immunofluorescence microscopy, we demonstrated that C. merolae contains one to two Golgi bodies per cell. The Golgi body was localized to the perinuclear region during the G1 and S phases and next to the spindle poles in a microtubule-dependent manner during M phase. It was inherited together with spindle poles upon cytokinesis. These observations suggested that Golgi inheritance is dependent on microtubules in C. merolae.     

Transgenic zebrafish for ratiometric imaging of cytosolic and mitochondrial Ca response in teleost embryo

Intracellular Ca²⁺ imaging has widely been used to visualize intracellular signals, but the application in an intact animal is still limited due to difficulty of the indicator loading. In addition, the motion of the living animal produces artifacts. To investigate Ca²⁺ signaling at early embryonic stage, we established transgenic zebrafish line expressing a genetically encoded Ca²⁺ indicator, cameleon YC2.60, driven by a constitutively active promoter, hspa8. Although the embryo dynamically changes its morphology, the motion artifact could be canceled out by taking the advantage of YC2.60 as a ratiometric indicator. The transgenic zebrafish was used to visualize the propagation of cytosolic Ca²⁺ during the early embryonic stage upon fertilization and along cleavage furrow, and the rise in Ca²⁺ in the myocytes contracting spontaneously in the embryo. We also established a transgenic zebrafish line expressing YC2.60 targeted to the mitochondria. The rise in mitochondrial Ca²⁺ was rather sustained (≈2 min), which is consistent with the requirement of ATP refilling since the mitochondrial Ca²⁺ upregulates rate-limiting enzymes of Krebs cycle. This is in contrast with the transient rise in the cytosol Ca²⁺ that directly evokes the muscle contraction. These transgenic zebrafish lines are expected to serve as useful tools further Ca²⁺ imaging in vivo.