Types and development pathways of lateral line scales in some teleost species

Voronina, E.P. and Hughes, D.R. 2011. Types and development pathways of lateral line scales in some teleost species. —Acta Zoologica (Stockholm) 00 : 1–13. A comparative study of lateral line scales (lls) in nine teleost species was undertaken to trace their ontogenetic structural changes. Three universal characters were used to describe and classify definitive and developing lls. The four main structural types in teleosts are represented. In adult fish, lls are the same structural type in all parts of lateral line in any one specimen, but number of tubules and their orientation may vary. In juvenile fish, except for one species, the structural type of every lls changes with growth, and this process progresses along the lateral line in the direction of development typical for the species. Definitive structural type of the lls is not determined by common scale type and size, presence or absence of nerve foramen on lls, scale overlapping or time of initiation of scales and trunk canal. Development pathways are proposed in which terminal states correspond to the final development of the most complex lls type in Cyprinus carpio, Carassius carassius, Oncorhynchus mykiss, Diplodus annularis and Mullus barbatus. The intermediate states of these pathways correspond to other types of lls as examples of pedomorphosis in Perca fluviatilis, Sander lucioperca, Symphysodon aequifasciatus and Hippoglossoides platessoides.     

Feeding behaviour and performance of different populations of the black currant‐lettuce aphid,Nasonovia ribisnigri,on resistant and susceptible lettuce

When crops are bred for resistance to herbivores, these herbivores are under strong selection pressure to overcome this resistance, which may result in the emergence of virulent biotypes. This is a growing problem for crop species attacked by aphids. The Nr‐gene in lettuce confers near‐complete resistance against the black currant‐lettuce aphid, Nasonovia ribisnigri (Mosely) (Hemiptera: Aphididae). Since 2007, populations of N. ribisnigri have been reported in several locations in Europe to infest resistant lettuce varieties that possess the Nr‐gene. The objective of this study was to analyse the behaviour and level of virulence of several N. ribisnigri populations observed to have colonized Nr‐locus‐containing lettuce lines. We analysed the stylet penetration and feeding behaviour, and the performance of these N. ribisnigri populations on resistant and susceptible lettuce lines. Large variation in the degree of virulence to the Nr‐locus‐containing lettuce lines was found among populations of the Nr:1 biotype. The German population was highly virulent on the Nr‐containing resistant lettuce lines, and showed similar feeding behaviour and performance on both the susceptible and resistant lettuces. The French population from Paris was the second most virulent, though reproduction on the resistant lines was reduced. The French population from Perpignan and a population from Belgium, however, showed reduced performance and feeding rate on the resistant compared to the susceptible lettuces. The lettuce background in which the Nr‐gene is expressed influences the level of resistance to the various Nr:1 aphid populations, because the performance and feeding behaviour differed between the aphids on the cultivars (romaine lettuce) compared to the near‐isogenic lines (butterhead/iceberg lettuce). This study also shows that being able to feed on a plant not automatically implies that a population can successfully develop on that plant, because aphids showed phloem ingestion during the 8‐h recording period on resistant lettuce, but were not able to survive and reproduce on the same lettuce line.     

Calpain2 protease: A new member of the Wnt/Ca pathway modulating convergent extension movements in Xenopus

Calpains are a family of calcium-dependent intracellular cysteine proteases that regulate several physiological processes by limited cleavage of different substrates. The role of Calpain2 in embryogenesis is not clear with conflicting evidence from a number of mouse knockouts. Here we report the temporal and spatial expression of Calpain2 in Xenopus laevis embryos and address its role in Xenopus development. We show that Calpain2 is expressed maternally with elevated expression in neural tissues and that Calpain2 activity is spatially and temporally regulated. Using a Calpain inhibitor, a dominant negative and a morpholino oligonoucleotide we demonstrate that impaired Calpain2 activity results in defective convergent extension both in mesodermal and neural tissues. Specifically, Calpain2 downregulation results in loss of tissue polarity and blockage of mediolateral intercalation in Keller explants without affecting adherens junction turnover. We further show that Calpain2 is activated in response to Wnt5a and that the inhibitory effect of Wnt5a expression on animal cap elongation can be rescued by blocking Calpain2 function. This suggests that Calpain2 activity needs to be tightly regulated during convergent extension. Finally we show that expression of Xdd1 blocks the membrane translocation of Calpain2 suggesting that Calpain2 activation is downstream of Dishevelled. Overall our data show that Calpain2 activation through the Wnt/Ca²⁺ pathway and Dishevelled can modulate convergent extension movements.     

Development of a highly-efficient CHO cell line generation system with engineered SV40E promoter

Chinese hamster ovary (CHO) cells have been one of the most widely used host cells for the manufacture of therapeutic recombinant proteins. An effective and efficient clinical cell line development process, which could quickly identify those rare, high-producing cell lines among a large population of low and non-productive cells, is of considerable interest to speed up biological drug development. In the glutamine synthetase (GS)-CHO expression system, selection of top-producing cell lines is based on controlling the balance between the expression level of GS and the concentration of its specific inhibitor, l-methionine sulfoximine (MSX). The combined amount of GS expressed from plasmids that have been introduced through transfection and the endogenous CHO GS gene determine the stringency and efficiency of selection. Previous studies have shown significant improvement in selection stringency by using GS-knockout CHO cells, which eliminate background GS expression from the endogenous GS gene in CHOK1SV cells. To further improve selection stringency, a series of weakened SV40E promoters have been generated and used to modulate plasmid-based GS expression with the intent of manipulating GS-CHO selection, finely adjusting the balance between GS expression and GS inhibitor (MSX) levels. The reduction of SV40E promoter activities have been confirmed by TaqMan RT-PCR and GFP expression profiling. Significant productivity improvements in both bulk culture and individual clonal cell line have been achieved with the combined use of GS-knockout CHOK1SV cells and weakened SV40E promoters driving GS expression in the current cell line generation process. The selection stringency was significantly increased, as indicated by the shift towards higher distribution of producing-cell populations, even with no MSX added into cell culture medium. The potential applications of weakened SV40E promoter and GS-knockout cells in development of targeted integration and transient CHO expression systems are also discussed.     

Gordonan,an Acidic Polysaccharide with Cell Aggregation-Inducing Activity in Insect BM-N4 Cells,Produced by Gordonia sp.

An acidic polysaccharide, termed gordonan, was isolated from the culture medium of Gordonia sp. as an inducer of cell aggregation in an insect cell line, BM-N4. Gordonan had an average molecular weight of 5×10⁶ and its structure was identified as →3)-4-O-(1-carboxyethyl)-β-D-Manp-(1→4)-β-D-GlcAp-(1→4)-β-D-Glcp-(1→ mainly by acid hydrolysis experiments and NMR analysis. It induces cell aggregation at the concentration of 4 μg/ml. A partially hydrolyzed polysaccharide derived from gordonan with a molecular weight of 5×10⁵ showed weak activity, while any fragment molecules with lower molecular weights prepared from gordonan showed no activity.     

The Purification and the Properties of Three Kinds of Lipases from Rhizopus delemer

The study was undertaken to clarify whether three kinds of lipases (EC 3.1.1.3) secreted from Rhizopus delemar are originally different or identical with each other. First of all, the purification of those lipases was carried out and their enzymatic properties were examined. Their properties including the stability on heat and pH, precipitabilities at a certain pH, the behaviours on a SE-Sephadex C50 column and on a Sephadex G200 column and so on were compared.

From the results, A-lipase is clearly different from the other two lipases. On the other hand, it seems that B- and C-lipases are originally identical.     

Biosynthesis of linoleic acid in Tyrophagus mites (Acarina: Acaridae)

We report here that Tyrophagus similis and Tyrophagus putrescentiae (Astigmata: Acaridae) have the ability to biosynthesize linoleic acid [(9Z, 12Z)-9, 12-octadecadienoic acid] via a Δ12-desaturation step, although animals in general and vertebrates in particular appear to lack this ability. When the mites were fed on dried yeast enriched with d₃₁-hexadecanoic acid (16:0), d₂₇-octadecadienoic acid (18:2), produced from d₃₁-hexadecanoic acid through elongation and desaturation reactions, was identified as a major fatty acid component of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in the mites. The double bond position of d₂₇-octadecadienoic acid (18:2) of PCs and PEs was determined to be 9 and 12, respectively by dimethyldisulfide (DMDS) derivatization. Furthermore, the GC/MS retention time of methyl 9, 12-octadecadienoate obtained from mite extracts agreed well with those of authentic linoleic acid methyl ester. It is still unclear whether the mites themselves or symbiotic microorganisms are responsible for inserting a double bond into the Δ12 position of octadecanoic acid. However, we present here the unique metabolism of fatty acids in the mites.     

THE EFFECTS OF BACK EXTENSION TRAINING ON BACK MUSCLE STRENGTH AND SPINAL RANGE OF MOTION IN YOUNG FEMALES

The objective of this study was to determine the effects of a 10-week dynamic back extension training programme and its effects on back muscle strength, back muscle endurance and spinal range of motion (ROM) for healthy young females. Seventy-three young females (age: 19.32±1.80 years, height: 158.89±4.71 cm, body weight: 55.67±6.30 kg) volunteered for the study. Prior to the training period, all participants completed anthropometric measurements, back muscle strength and endurance test, lateral bending and spinal ROM measurements. After measurements, the participants were divided into two groups. The exercise group (N:35) performed the dynamic back extension exercise 3 days per week for 10 weeks. The control group (N:38) did not participate in any type of exercise. The mixed design ANOVA (group x time) was used to determine the difference in pre- and post-training values. The present findings show that there were significant differences between pre-training and post-training values for back muscle strength and spinal ROM in the exercise group. Following the dynamic strength training programme, back muscle strength and spine ROM values except flexion of the lumbar 5-sacrum 1 (L5-S1) segment of the exercise group showed a significant increase when compared with the pre test values. The control group did not show any significant changes when compared with the pre-training values. The results demonstrate that the 10-week dynamic strength training programme was effective for spinal extension ROM and back muscle strength, but there was no change in back muscle endurance. In this context, this programme could potentially be used to prevent the decrease of spinal ROM as well as provide an increase in the fitness parameters of healthy individuals.     

A deleterious effect associated with UNH159 is attenuated in twin embryos of an inbred line of blue tilapia Oreochromis aureus

Offspring of a highly inbred gynogenetic line of Oreochromis aureus displayed 12‐fold increase in twinning rate compared to the outbred population. Asymmetric conjoined twins, which consist of a normal embryo attached to a malformed‐atrophic twin, were frequently encountered in both gynogenetic (90·7%) and outbred (38·2%) embryos. The monozygotic origin of these twins was determined using five microsatellite markers. Progeny of heterozygous parents for the microsatellite UNH159 were separated into sub‐sets of twins and normal full‐sibs. Consistent with previous reports, the normal embryo sub‐set exhibited elimination of both types of homozygotes for the UNH159 genetic marker at 2–8 days after fertilization. Unexpectedly, this elimination was less frequent in twins. The UNH159 marker as well as RNA‐binding motif protein, X‐linked (rbmx), SRY‐box containing gene 3 (sox3) and alpha‐thalassemia/mental retardation syndrome X‐linked (atrx) genes were mapped to linkage group 2. These gene orthologues are all located on the mammalian X chromosome and atrx is necessary for the X‐chromosome inactivation.     

Subtractive phage display technology identifies zebrafish marcksb that is required for gastrulation

In the present study, we used a phage display technique to screen differentially expressed proteins from zebrafish post-gastrula embryos. With a subtractive screening approach, 6 types of single-chain Fv fragments (scFvs) were screened out from an scFv antibody phage display library by biopanning against zebrafish embryonic homogenate. Four scFv fragments (scFv1, scFv3, scFv4 and scFv6) showed significantly stronger binding to the tailbud embryos than to the 30%-epiboly embryos. A T7 phage display cDNA library was constructed from zebrafish tailbud embryos and used to identify the antigens potentially recognized by scFv1, which showed the highest frequency and strongest binding against the tailbud embryos. We acquired 4 candidate epitopes using scFv1 and the corresponding genes showed significantly higher expression levels at tailbud stage than at 30%-epiboly. The most potent epitope of scFv1 was the clone scFv1-2, which showed strong homology to zebrafish myristoylated alanine-rich C-kinase substrate b (Marcksb). Western blot analysis confirmed the high expression of marcksb in the post-gastrula embryos, and the endogenous expression of Marcksb was interfered by injection of scFv1. Zebrafish marcksb showed dynamic expression patterns during embryonic development. Knockdown of marcksb strongly affected gastrulation movements. Moreover, we revealed that zebrafish marcksb is required for cell membrane protrusion and F-actin alignment. Thus, our study uncovered 4 types of scFvs binding to zebrafish post-gastrula embryos, and the epitope of scFv1 was found to be required for normal gastrulation of zebrafish. To our knowledge, this was the first attempt to combine phage display technique with the embryonic and developmental study of vertebrates, and we were able to identify zebrafish marcksb that was required for gastrulation.