Rapid directed molecular evolution of fluorescent proteins in mammalian cells

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2 × 10⁷ independent random genes of fluorescent proteins expressed in HEK cells, completing one iteration of directed evolution in a course of 8 days. We employed this approach to develop a set of green and near‐infrared fluorescent proteins with enhanced intracellular brightness. The developed near‐infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as Caenorhabditis elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near‐infrared fluorescent proteins enabled crosstalk‐free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual‐color near‐infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multicolor imaging of small model organisms.     

Hemagglutinating and adherence properties of Staphylococcus saprophyticus: epidemiology and virulence in experimental urinary tract infection of rats

Abstract We studied hemagglutinating and adherence properties in Staphylococcus saprophyticus isolates originating from symptomatic urinary tract infections. 12/13 (92%) of strains adhered to Hep cells and 11/13 (85%) were able to agglutinate sheep erythrocytes. Adherence properties differed markedly between strains ( P < 0.0001. Two strains, one hemagglutinating and adherent and one negative for both properties were chosen for experimental urinary tract infections. Results indicate that presence of the hemagglutinin favours colonization of kidney tissue.     

Changes in the auditory neuropil after deafferentation in adult grasshoppers (Schistocerca gregaria)

Nervous systems are capable of structural adjustments. Such plastic changes also occur in the auditory system of the locust Schistocerca gregaria in which a deafferentation leads to compensatory mechanisms, such as collateral sprouting of interneurons. In this study we further investigated lesion related changes in the major auditory neuropil, the median ventral association center (mVAC) of the metathoracic ganglion. The auditory sensory organ of adult locusts was unilaterally extirpated and the mVAC was histologically and immunocytochemically analyzed until 20 days postoperative. Measurements of the neuropil area in transverse sections showed a decrease in size. The putative transmitter of the afferents, acetylcholine, was investigated by acetylcholinesterase histochemistry. Comparisons of staining intensities in the intact and deafferentated mVAC indicated that the amount of acetylcholinesterase in the deafferentated mVAC decreased shortly after the operation. Both, the decreases in size of the mVAC as well as that in acetylcholinesterase histochemistry were only less than 10% compared to the controls. The immunoreactivity against the neurotransmitters γ-amino butyric acid and serotonin was not influenced by the deafferentation.     

beta-thymosin is required for axonal tract formation in developing zebrafish brain

beta-Thymosins are polypeptides that bind monomeric actin and thereby function as actin buffers in many cells. We show that during zebrafish development, &bgr;-thymosin expression is tightly correlated with neuronal growth and differentiation. It is transiently expressed in a subset of axon-extending neurons, essentially primary neurons that extend long axons, glia and muscle. Non-neuronal expression in the brain is restricted to a subset of glia surrounding newly forming axonal tracts. Skeletal muscle cells in somites, jaw and fin express beta-thymosin during differentiation, coinciding with the time of innervation. Injection of beta-thymosin antisense RNA into zebrafish embryos results in brain defects and impairment of the development of beta-thymosin-associated axon tracts. Furthermore, irregularities in somite formation can be seen in a subset of embryos. Compared to wild-type, antisense-injected embryos show slightly weaker and more diffuse engrailed staining at the midbrain-hindbrain boundary and a strong reduction of Isl-1 labeling in Rohon Beard and trigeminal neurons. The decreased expression is not based on a loss of neurons indicating that beta-thymosin may be involved in the maintenance of the expression of molecules necessary for neuronal differentiation. Taken together, our results strongly indicate that beta-thymosin is an important regulator of development.     

Placental failure and impaired vasculogenesis result in embryonic lethality for neuropathy target esterase-deficient mice

Age-dependent neurodegeneration resulting from widespread apoptosis of neurons and glia characterize the Drosophila Swiss Cheese (SWS) mutant. Neuropathy target esterase (NTE), the vertebrate homologue of SWS, reacts with organophosphates which initiate a syndrome of axonal degeneration. NTE is expressed in neurons and a variety of nonneuronal cell types in adults and fetal mice. To investigate the physiological functions of NTE, we inactivated its gene by targeted mutagenesis in embryonic stem cells. Heterozygous NTE(+/-) mice displayed a 50% reduction in NTE activity but underwent normal organ development. Complete inactivation of the NTE gene resulted in embryonic lethality, which became evident after gastrulation at embryonic day 9 postcoitum (E9). As early as E7.5, mutant embryos revealed growth retardation which did not reflect impaired cell proliferation but rather resulted from failed placental development; as a consequence, massive apoptosis within the developing embryo preceded its resorption. Histological analysis indicated that NTE is essential for the formation of the labyrinth layer and survival and differentiation of secondary giant cells. Additionally, impairment of vasculogenesis in the yolk sacs and embryos of null mutant conceptuses suggested that NTE is also required for normal blood vessel development.     

The Sp8 zinc-finger transcription factor is involved in allometric growth of the limbs in the beetle Tribolium castaneum

Members of the Sp gene family are involved in a variety of developmental processes in both vertebrates and invertebrates. We identified the ortholog of the Drosophila Sp-1 gene in the red flour beetle Tribolium castaneum, termed T-Sp8 because of its close phylogenetic relationship to the vertebrate Sp8 genes. During early embryogenesis, T-Sp8 is seen in segmental stripes. During later stages, TSp8 is dynamically expressed in the limb buds of the Tribolium embryo. At the beginning of bud formation, TSp8 is uniformly expressed in all body appendages. As the limbs elongate, a ring pattern develops sequentially and the expression profile at the end of embryogenesis correlates with the final length of the appendage. In limbs that do not grow out like the labrum and the labium, T-Sp8 expression remains uniform, whereas a two-ring pattern develops in the longer antennae and the maxillae. In the legs that elongate even further, four rings of T-Sp8 expression can be seen at the end of leg development. The role of T-Sp8 for appendage development was tested using RNAi. Upon injection of double stranded T-Sp8 RNA, larvae develop with dwarfed appendages. Affected T-Sp8(RNAi) legs were tested for the presence of medial and distal positional values using the expression marker genes dachshund and Distal-less, respectively. The results show that a dwarfed TSp8(RNAi) leg consists of proximal, medial and distal parts and argues against T-Sp8 being a leg gap gene. Based on the differential expression pattern of T-Sp8 in the appendages of the head and the thorax and the RNAi phenotype, we hypothesise that T-Sp8 is involved in the regulation of limb-length in relation to body size - a process called allometric growth.     

Developmental origin of wiring specificity in the olfactory system of Drosophila

In both insects and mammals, olfactory receptor neurons (ORNs) expressing specific olfactory receptors converge their axons onto specific glomeruli, creating a spatial map in the brain. We have previously shown that second order projection neurons (PNs) in Drosophila are prespecified by lineage and birth order to send their dendrites to one of approximately 50 glomeruli in the antennal lobe. How can a given class of ORN axons match up with a given class of PN dendrites? Here, we examine the cellular and developmental events that lead to this wiring specificity. We find that, before ORN axon arrival, PN dendrites have already created a prototypic map that resembles the adult glomerular map, by virtue of their selective dendritic localization. Positional cues that create this prototypic dendritic map do not appear to be either from the residual larval olfactory system or from glial processes within the antennal lobe. We propose instead that this prototypic map might originate from both patterning information external to the developing antennal lobe and interactions among PN dendrites.     

Amperometric determination of lactate with novel trienzyme/poly(carbamoyl) sulfonate hydrogel-based sensor

A novel trienzyme sensor for the amperometric determination of lactate was constructed by immobilizing salicylate hydroxylase (SHL, E.C. 1.14.13.1), l-lactate dehydrogenase (LDH, E.C. 1.1.1.27), and pyruvate oxidase (PyOD, E.C. 1.2.3.3) on a Clark-type oxygen electrode. The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. LDH catalyzes the specific dehydrogenation of lactate consuming NAD(+). SHL catalyzes the irreversible decarboxylation and the hydroxylation of salicylate in the presence of oxygen and NADH produced by LDH. PyOD decarboxylates pyruvate using oxygen and phosphate. SHL and PyOD force the equilibrium of dehydrogenation of lactate by LDH to the product side by consuming NADH and pyruvate, respectively. Dissolved oxygen acts as an essential material for both PyOD and SHL during their respective enzymatic reactions. Therefore, an amplified signal, caused by the consumptions of dissolved oxygen by the two enzymes, was observed in the measurement of lactate. Regeneration of cofactor was found in the trienzyme system. A Teflon membrane was used to fabricate the sensor in order to avoid interferences. The sensor has a fast response (2s) and short recovery times (2 min). The total test time for a measurement by using this lactate sensor (4 min) was faster than using a commercial lactate testing kit (up to 10 min). The sensor has a linear range between 10 and 400 microM lactate, with a detection limit of 4.3 microM. A good agreement (R2 = 0.9984) with a commercial lactate testing kit was obtained in beverage sample measurements.     

Purification and characterization of 9-O-acetylated sialoglycoproteins from leukemic cells and their potential as immunological tool for monitoring childhood acute lymphoblastic leukemia

Sialic acids as terminal residues of oligosaccharide chains play crucial roles in several cellular recognition events. Exploiting the selective affinity of Achatinin-H toward N-acetyl-9-O-acetylneuraminic acid-alpha2-6-GalNAc, we have demonstrated the presence of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) on lymphoblasts of 70 children with acute lymphoblastic leukemia (ALL) and on leukemic cell lines by fluorimetric HPLC and flow cytometric analysis. This study aims to assess the structural aspect of the glycotope of Neu5,9Ac(2)-GPs(ALL) and to evaluate whether these disease-specific molecules can be used to monitor the clinical outcome of ALL. The Neu5,9Ac(2)-GPs(ALL) were affinity-purified, and three distinct leukemia-specific molecular determinants (135, 120, and 90 kDa) were demonstrated by SDS-PAGE, western blotting, and isoelectric focusing. The carbohydrate epitope of Neu5,9Ac(2)-GPs(ALL) was confirmed by using synthetic sialic acid analogs. The enhanced presence of anti-Neu5,9Ac(2)-GP(ALL) antibody in ALL patients prompted us to develop an antigen-ELISA using purified Neu5,9Ac(2)-GPs(ALL) as coating antigens. Purified antigen was able to detect leukemia-specific antibodies at presentation of disease, which gradually decreased with treatment. Longitudinal monitoring of 18 patients revealed that in the early phase of the treatment patients with lower anti-Neu5,9Ac(2)-GPs showed a better prognosis. Minimal cross-reactivity was observed in other hematological disorders (n = 50) like chronic myeloid leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma as well as normal healthy individuals (n = 21). This study demonstrated the potential of purified Neu5,9Ac(2)-GPs(ALL) as an alternate tool for detection of anti-Neu5,9Ac(2)-GP antibodies to be helpful for diagnosis and monitoring of childhood ALL patients.