Functional characterization of the zebrafish WHSC1-related gene, a homolog of human NSD2.

Methylation of specific lysine residues of histone H3 and H4 has been reported to be important in the structuring of chromatin and for the transcription of certain genes. Proteins with SET domains have been shown to methylate specific lysine residues of histone H3 and H4. We isolated a SET domain-containing gene from the zebrafish (Danio rerio). The gene has the highest sequence similarity to human NSD2, also known as Wolf-Hirschhorn syndrome candidate 1 or WHSC1, and therefore, was named DrWhsc1. DrWhsc1 mRNA is expressed in various tissues with the highest level in testis. Morpholino oligonucleotides for the DrWhsc1 gene affected early embryogenesis in zebrafish, such as endbrain enlargement, abnormal cartilage, marked reduction of bone, and incomplete motor neuron formation. Such developmental abnormalities are also observed in Wolf-Hirschhorn syndrome patients and Whsc1-deficient mice. In addition, suppression of the DrWhsc1 gene or defect in the SET domain of DrWhsc1 resulted in impairment of di-methylation of histone H3K36 at early embryogenesis. These results indicate that DrWhsc1 is a functional homolog of WHSC1 and that the SET domain of DrWhsc1 is essential for di-methylation of histone H3K36 in zebrafish.

Preparation and Purification of Active Recombinant Human Pancreatic Lipase in Escherichia coli.

Human pancreatic lipase (HPL) is the main lipolytic enzyme involved in the digestion of dietary fat. An active recombinant human pancreatic lipase (recHPL) was successfully prepared for the first time in an Escherichia coli (E. coli) expression system using a short Strep-tag II (ST II). The recHPL-ST II was solubilized with 8 M urea from the E. coli lysate and purified on a Strep-Tactin-Sepharose column. After refolding by stepwise dialyses against decreasing concentrations of urea in the presence of glycerol and Ca2+ for two days followed by gel filtration FPLC, 1.8-6 mg of active recHPL-ST II was obtained from 1 L of culture. Here we report the expression, purification, and optimized refolding procedures for active recHPL from E. coli, thus establishing it as a suitable system for the production of recHPL of high purity and scaling up.

A novel protocol for the preparation of active recombinant human pancreatic lipase from Escherichia coli.

An active recombinant human pancreatic lipase (recHPL) was successfully prepared for the first time from the Escherichia coli expression system using short Strep-tag II (ST II). The recHPL-ST II was solubilized using 8 M urea from E.coli lysate and purified on a Strep-Tactin-Sepharose column. After refolding by stepwise dialyses in the presence of glycerol and Ca2+ for 2 days followed by gel filtration, 1.8-6 mg of active recHPL-ST II was obtained from 1 L of culture. The recHPL was non-glycosylated, but showed almost equal specific activity, pH-dependency and time-dependent stability compared to those of native porcine pancreatic lipase (PPL) at 37°C. However, the recHPL lost its lipolytic activity above 50°C, showing a lower heat-stability than that of native PPL, which retained half its activity at this temperature.

Ontogenic development of three GnRH systems in the brain of a pleuronectiform fish, barfin flounder.

A pleuronectiform fish, the barfin flounder Verasper moseri, has three molecular forms of gonadotropin-releasing hormone (GnRH) in the brain, salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and seabream GnRH (sbGnRH). To elucidate the ontogenic origin of the neurons that produce these GnRH molecules, the development of three GnRH systems was examined by in situ hybridization and immunocytochemistry. Neuronal somata that express sGnRH mRNA were detected first in the vicinity of the olfactory epithelium 21 days after hatching (Day 21), and then in the transitional area between the olfactory nerve and olfactory bulb and the terminal nerve ganglion on Day 28. cGnRH-II mRNA-expressing neuronal somata were first identified in the midbrain tegmentum near the ventricle on Day 7. cGnRH-II-immunoreactive (ir) fibers were first found in the brain on Day 7. sbGnRH mRNA-expressing neuronal somata were first detected in the preoptic area on Day 42. sbGnRH-ir fibers were localized in the preoptic area-hypothalamus, and formed a distinctive bundle of axons projecting to the pituitary on Day 70. These results indicate that three forms of GnRH neurons have separate embryonic origins in the barfin flounder as in other perciform fish such as tilapia Oreochromis niloticus and red seabream Pagrus major: sGnRH, cGnRH-II and sbGnRH neurons derive from the olfactory placode, the midbrain tegmentum near the ventricle and the preoptic area, respectively.