Fine mapping of the circling (cir) gene on the distal portion of mouse chromosome 9

Circling mice manifest profound deafness, head-tossing, and bi-directional circling behavior, which they inherit in autosomal recessive manner. Histologic examination of the inner ear reveals abnormalities of the region around the organ of Corti, spiral ganglion neurons, and outer hair cells. A genetic linkage map was constructed for an intraspecific backcross between cir and C57BL/6J mice. The cir gene was mapped to a region between D9Mit116/D9Mit15 and D9Mit38 on mouse chromosome (Chr) 9. Estimated distances between cir and D9Mit116, and between cir and D9Mit38 were 0.70 +/- 0.40 and 0.23 +/- 0.23 cM, respectively. Order of the markers was defined as follows: centromere - D9Mit182 - D9Mit51/D9Mit79/D9Mit310 - D9Mit212/D184 - D9Mit116/D9Mit15 - cir - D9Mit38 - D9Mit20 - D9Mit243 - D9Mit16 - D9Mit55/D9Mit125 - D9Mit281. On the basis of genetic mapping, we constructed a yeast artificial chromosome (YAC) contig across the cir region. The cir gene is located between the lactotransferrin (ltf) and microtubule-associated protein (map4) genes. The distal portion of mouse Chr 9 encompassing the cir region is homologous with human chromosome 3p21, which contains the Deafness, form B: Autosomal Recessive Deafness (DFNB6) locus. Therefore, the circling mouse is a potential animal model for DFNB6 deafness in humans.     

Rice proteins that bind single-stranded G-rich telomere DNA

In this work, we have identified and characterized proteins in rice nuclear extracts that specifically bind the single-stranded G-rich telomere sequence. Three types of specific DNA-protein complexes (I, II, and III) were identified by gel retardation assays using synthetic telomere substrates consisting of two or more single-stranded TTTAGGG repeats and rice nuclear extracts. Since each complex has a unique biochemical property and differs in electrophoretic mobility, at least three different proteins interact with the G-rich telomere sequences. These proteins are called rice G-rich telomere binding protein (RGBP) and none of them show binding affinity to double-stranded telomere repeats or single-stranded C-rich sequence. Changing one or two G's to C's in the TTTAGGG repeats abolishes binding activity. RGBPs have a greatly reduced affinity for human and Tetrahymena telomeric sequence and do not efficiently bind the cognate G-rich telomere RNA sequence UUUAGGG. Like other telomere binding proteins, RGBPs are resistant to high salt concentrations. RNase sensitivity of the DNA-protein interactions was tested to investigate whether an RNA component mediates the telomeric DNA-protein interaction. In this assay, we observed a novel complex (complex III) in gel retardation assays which did not alter the mobilities or the band intensities of the two pre-existing complexes (I and II). The complex III, in addition to binding to telomeric sequences, has a binding affinity to rice nuclear RNA, whereas two other complexes have a binding affinity to only single-stranded G-rich telomere DNA. Taken together, these studies suggest that RGBPs are new types of telomere-binding proteins that bind in vitro to single-stranded G-rich telomere DNA in the angiosperms.     

Use of Composite Protein Database including Search Result Sequences for Mass Spectrometric Analysis of Cell Secretome

Mass spectrometric (MS) data of human cell secretomes are usually run through the conventional human database for identification. However, the search may result in false identifications due to contamination of the secretome with fetal bovine serum (FBS) proteins. To overcome this challenge, here we provide a composite protein database including human as well as 199 FBS protein sequences for MS data search of human cell secretomes. Searching against the human-FBS database returned more reliable results with fewer false-positive and false-negative identifications compared to using either a human only database or a human-bovine database. Furthermore, the improved results validated our strategy without complex experiments like SILAC. We expect our strategy to improve the accuracy of human secreted protein identification and to also add value for general use.     

Does the Mother or Father Determine the Offspring Sex Ratio? Investigating the Relationship between Maternal Digit Ratio and Offspring Sex Ratio

Objective

In mammals, high parental testosterone levels present around the time of conception are thought to skew offspring sex ratio toward sons. The second to fourth digit ratio (digit ratio) is now widely accepted as a negative correlate of prenatal testosterone. Thus, we investigated the association between digit ratio and offspring sex ratio.

Methods

A total of 508 Korean patients (257 males and 251 females) less than 60 years old who had one or more offspring were prospectively enrolled. The lengths of the 2nd and 4th digits of the right hand were measured by a single investigator using a digital vernier calliper. Next, the patients’ lifetime offspring birth sex ratios were investigated.

Results

Maternal (rather than paternal) digit ratio was significantly associated with the number of sons (r = -0.153, p = 0.015), number of daughters (r = 0.130, p = 0.039), and offspring sex ratio (r = -0.171, p = 0.007). And, the maternal digit ratio was a significant factor for predicting offspring sex ratio (B = -1.620, p = 0.008) on multiple linear regression analysis. The female patients with a lower digit ratio (< 0.95) were found to have a higher offspring sex ratio (0.609 versus 0.521, p = 0.046) compared to those with a higher digit ratio (≥ 0.95). Furthermore, females in the low digit ratio group have a probability 1.138 greater of having sons than females in the high digit ratio group.

Conclusions

Maternal digit ratio was negatively associated with offspring sex ratio. Females with a lower digit ratio were more likely to have more male offspring compared to those with a higher digit ratio. Thus, our results suggest that the sex of offspring might be more influenced by maternal rather than paternal factors.     

Neurotropic and neuroprotective activities of the earthworm peptide Lumbricusin

We recently isolated a polypeptide from the earthworm Lumbricus terrestris that is structurally similar to defensin, a well-known antibacterial peptide. An 11-mer antibacterial peptide (NH₂-RNRRWCIDQQA), designated Lumbricusin, was synthesized based on the amino acid sequence of the isolated polypeptide. Since we previously reported that CopA3, a dung beetle peptide, enhanced neuronal cell proliferation, we here examined whether Lumbricusin exerted neurotropic and/or neuroprotective effects. Lumbricusin treatment induced a time-dependent increase (∼51%) in the proliferation of human neuroblastoma SH-SY5Y cells. Lumbricusin also significantly inhibited the apoptosis and decreased viability induced by treatment with 6-hydroxy dopamine, a Parkinson’s disease-mimicking agent. Immunoblot analyses revealed that Lumbricusin treatment increased ubiquitination of p27^Kip1 protein, a negative regulator of cell-cycle progression, in SH-SY5Y cells, and markedly promoted its degradation. Notably, adenoviral-mediated over-expression of p27^Kip1 significantly blocked the antiapoptotic effect of Lumbricusin in 6-hydroxy dopamine-treated SH-SY5Y cells. These results suggest that promotion of p27^Kip1 degradation may be the main mechanism underlying the neuroprotective and neurotropic effects of Lumbricusin.     

Solution structure of CEH-37 homeodomain of the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans protein CEH-37 belongs to the paired OTD/OTX family of homeobox-containing homeodomain proteins. CEH-37 shares sequence similarity with homeodomain proteins, although it specifically binds to double-stranded C. elegans telomeric DNA, which is unusual to homeodomain proteins. Here, we report the solution structure of CEH-37 homeodomain and molecular interaction with double-stranded C. elegans telomeric DNA using nuclear magnetic resonance (NMR) spectroscopy. NMR structure shows that CEH-37 homeodomain is composed of a flexible N-terminal region and three α-helices with a helix-turn-helix (HTH) DNA binding motif. Data from size-exclusion chromatography and fluorescence spectroscopy reveal that CEH-37 homeodomain interacts strongly with double-stranded C. elegans telomeric DNA. NMR titration experiments identified residues responsible for specific binding to nematode double-stranded telomeric DNA. These results suggest that C. elegans homeodomain protein, CEH-37 could play an important role in telomere function via DNA binding.     

Biological attributes of Ooencyrtus nezarae Ishii (Hymenoptera: Encyrtidae) reared on refrigerated eggs of Riptortus pedestris (= clavatus) Fabricius (Hemiptera: Alydidae)

Storing host eggs at low temperatures has been used to mass rear parasitoids of stink bugs, including Riptortus pedestris Fabricius (Hemiptera: Alydidae), a major soybean pest in Korea and Japan. However, no information on the effect of cold storage on parasitization by Ooencyrtus nezarae Ishii (Hymenoptera: Encyrtidae), one of the major parasitoids of R. pedestris, has been published. In this study, we examined biological attributes, including parasitism rate, development time, sex ratio, adult size, and longevity, of O. nezarae when the adult parasitoids were provided with host eggs refrigerated for 0, 15, 30, 60, and 120 days at 2.0 °C. None of the attributes of the first or second generation of O. nezarae was negatively affected by host egg refrigeration up to 30 days. In addition, O. nezarae could parasitize refrigerated host eggs successfully for the first four days of post-refrigeration period when they were kept at 26.3 °C and 78.7% RH conditions. Therefore, refrigeration of R. pedestris eggs can be a good method to mass rear O. nezarae.     

Isolation and characterization of two Korean mistletoe lectins

Two isolectins (KML-IIU and the KML-IIL) were individually isolated from the previously reported Korean mistletoe lectin, KML-C, by using an immunoaffinity column. Molecular weights of the KML-IIU and the KML-IIL were 64 kDa and 60 kDa respectively. Both of the lectins were composed of heterogeneous A and B subunits linked with a disulfide bond, and showed the same carbohydrate-binding specificities for Gal and GalNAc. However, they are different not only in biophysical properties (glycosylation and amino acid compositions) but also bioactivities (cell killing and cytokine induction). The KML-IIL showed 17-145 times stronger in cytotoxicities to various human and mouse cancer cell lines than the KML-IIU. The KML-IIL also induced TNF-alpha secretion from mouse peritoneal macrophages 4.5 times better than the KML-IIU. The results demonstrated isolectins in Korean mistletoe were varied in bioactivities and the KML-IIL may be developed as an anti-cancer agent.