Microsatellite DNA markers for population-genetic studies of blue mackerel (Scomber australasicus) and cross-specific amplification in S. japonicus

Blue mackerel (Scomber australasicus) is targeted by large-scale purse-seiners in the western North Pacific, and its stock structure is still contentious. Herein, we described 10 polymorphic microsatellite loci for blue mackerel. The number of alleles among 32 individuals surveyed ranged from five to 27 (average of 16.2 alleles per locus). Departures from Hardy-Weinberg expectation were observed at two loci. Cross-specific amplification in the congener, S. japonicus, was successful, except for one locus, revealed to be diagnostic for these congeners. These microsatellite loci will be useful tools to address queries in population genetic structure, fishery management unit and taxonomic species status in the genus Scomber.     

MicroRNA-138 Suppresses Neutrophil Gelatinase-Associated Lipocalin Expression and Inhibits Tumorigenicity

The expression of neutrophil gelatinase-associated lipocalin (NGAL) is up-regulated in some cancers; therefore NGAL has potential as a tumor biomarker. Although the regulation mechanism for this is unknown, one study has shown that it is likely to involve a microRNA (miRNA). Here, we investigate the relation between miRNA expression and NGAL expression, and the role of NGAL in tumorigenesis. Using miRNA target–detecting software, we analyze the mRNA sequence of NGAL and identify a target site for microRNA-138 (miR-138) in nucleotides 25–53 of the 3′ UTR. We then analyze NGAL and miR-138 expression in three cancer cell lines originating from breast, endometrial and pancreatic carcinomas (the MCF-7, RL95-2 and AsPC-1 cell lines), respectively, using quantitative (real-time) PCR and western blot analysis. Metastasis is a critical event in cancer progression, in which malignant cell proliferation, migration and invasion increase. To determine whether miR-138-regulated NGAL expression is associated with metastasis, the proliferation and migration of the cell line are examined after miR-138 transfection. Using nude mice, we examine both the tumorigenicity of these cell lines and of miR-138-transfected cancer cells in vivo, as well as the effect of treating tumors with an antibody against NGAL. Our results show that these cancer cell lines down-regulate NGAL when miR-138 is highly expressed. Ectopic transfection of miR-138 suppresses NGAL expression and cell migration in RL95-2 and AsPC-1 cells, demonstrating that miR-138-regulated NGAL expression is associated with cell migration. Additionally, injection of the NGAL antibody diminishes NGAL-mediated tumorigenesis in nude mice, and miR-138 transfection of cancer cells reduces tumor formation. As the cell proliferation data showed that the tumor size should be regulated by NGAL-related cell growth. Taken together, our results indicate that NGAL may be a good target for cancer therapy and suggest that miR-138 acts as a tumor suppressor and may prevent metastasis.     

Use of the Gram stain in microbiology

The Gram stain differentiates bacteria into two fundamental varieties of cells. Bacteria that retain the initial crystal violet stain (purple) are said to be 'Gram-positive,' whereas those that are decolorized and stain red with carbol fuchsin (or safranin) are said to be 'Gram-negative.' This staining response is based on the chemical and structural makeup of the cell walls of both varieties of bacteria. Gram-positives have a thick, relatively impermeable wall that resists decolorization and is composed of peptidoglycan and secondary polymers. Gram-negatives have a thin peptidoglycan layer plus an overlying lipid-protein bilayer known as the outer membrane, which can be disrupted by decolorization. Some bacteria have walls of intermediate structure and, although they are officially classified as Gram-positives because of their linage, they stain in a variable manner. One prokaryote domain, the Archaea, have such variability of wall structure that the Gram stain is not a useful differentiating tool.     

Protein dynamics and allostery: an NMR view

Allostery, the process by which distant sites within a protein system are energetically coupled, is an efficient and ubiquitous mechanism for activity regulation. A purely mechanical view of allostery invoking only structural changes has developed over the decades as the classical view of the phenomenon. However, a fast growing list of examples illustrate the intimate link between internal motions over a wide range of time scales and function in protein-ligand interactions. Proteins respond to perturbations by redistributing their motions and they use fluctuating conformational states for binding and conformational entropy as a carrier of allosteric energy to modulate association with ligands. In several cases allosteric interactions proceed with minimal or no structural changes. We discuss emerging paradigms for the central role of protein dynamics in allostery.     

Cloning of Two New cry Genes from Bacillus thuringiensis subsp. wuhanensis Strain

With PCR products as probes, we have cloned two new cry-type genes from Bacillus thuringiensis subsp. wuhanensis. The deduced amino acid sequence of the first clone is 77.3% identical to Cry1Ga1. The deduced protein sequence of the second clone is 69.8–78.7% identical to that of Cry1B group. The nomenclature assignment of these two clones is, therefore, named Cry1Gb1 and Cry1Bd1, respectively. The Cry1Bd1 is toxic to Plutella xylostella larvae, and the Cry1Gb1 is toxic to Pieris rapae larvae. Received: 2 August 1999 / Accepted: 18 October 1999     

The diageotropica mutation alters auxin induction of a subset of the Aux/IAA gene family in tomato

The diageotropica (dgt) mutation has been proposed to affect either auxin perception or responsiveness in tomato plants. It has previously been demonstrated that the expression of one member of the Aux/IAA family of auxin-regulated genes is reduced in dgt plants. Here, we report the cloning of ten new members of the tomato Aux/IAA family by PCR amplification based on conserved protein domains. All of the gene family members except one (LeIAA7) are expressed in etiolated tomato seedlings, although they demonstrate tissue specificity (e.g. increased expression in hypocotyls vs. roots) within the seedling. The wild-type auxin-response characteristics of the expression of these tomato LeIAA genes are similar to those previously described for Aux/IAA family members in Arabidopsis. In dgt seedlings, auxin stimulation of gene expression was reduced in only a subset of LeIAA genes (LeIAA5, 8, 10, and 11), with the greatest reduction associated with those genes with the strongest wild-type response to auxin. The remaining LeIAA genes tested exhibited essentially the same induction levels in response to the hormone in both dgt and wild-type hypocotyls. These results confirm that dgt plants can perceive auxin and suggest that a specific step in early auxin signal transduction is disrupted by the dgt mutation.     

Reduction in antioxidant enzyme expression and sustained inflammation enhance tissue damage in the subacute phase of spinal cord contusive injury

Background  

Traumatic spinal cord injury (SCI) forms a disadvantageous microenvironment for tissue repair at the lesion site. To consider an appropriate time window for giving a promising therapeutic treatment for subacute and chronic SCI, global changes of proteins in the injured center at the longer survival time points after SCI remains to be elucidated.     

Replication slippage may cause parallel evolution in the secondary structures of mitochondrial transfer RNAs

Presence of the dihydrouridine (D) stem in the mitochondrial cysteine tRNA is unusually variable among lepidosaurian reptiles. Phylogenetic and comparative analyses of cysteine tRNA gene sequences identify eight parallel losses of the D-stem, resulting in D-arm replacement loops. Sampling within the monophyletic Acrodonta provides no evidence for reversal. Slipped-strand mispairing of noncontiguous repeated sequences during replication or direct replication slippage can explain repeats observed within cysteine tRNAs that contain a D-arm replacement loop. These two mechanisms involving replication slippage can account for the loss of the cysteine tRNA D-stem in several lepidosaurian lineages, and may represent general mechanisms by which the secondary structures of mitochondrial tRNAs are altered.      

TLR2 and TLR4 Mediate Differential Responses to Limb Ischemia through MyD88-Dependent and Independent Pathways

Introduction

The danger signal HMGB1 is released from ischemic myocytes, and mediates angiogenesis in the setting of hindlimb ischemia. HMGB1 is a ligand for innate immune receptors TLR2 and TLR4. While both TLR2 and TLR4 signal through myeloid differentiation factor 88 (MyD88), TLR4 also uniquely signals through TIR-domain-containing adapter-inducing interferon-β (TRIF). We hypothesize that TLR2 and TLR4 mediate ischemic myocyte regeneration and angiogenesis in a manner that is dependent on MyD88 signaling.

Methods

Mice deficient in TLR2, TLR4, MyD88 and TRIF underwent femoral artery ligation in the right hindlimb. Laser Doppler perfusion imaging was used to assess the initial degree of ischemia and the extent of perfusion recovery. Muscle regeneration, necrosis and fat replacement at 2 weeks post-ligation were assessed histologically and vascular density was quantified by immunostaining. In vitro, endothelial tube formation was evaluated in matrigel in the setting of TLR2 and TLR4 antagonism.

Results

While control and TLR4 KO mice demonstrated prominent muscle regeneration, both TLR2 KO and TRIF KO mice exhibited marked necrosis with significant inflammatory cell infiltrate. However, MyD88 KO mice had a minimal response to the ischemic insult with little evidence of injury. This observation could not be explained by differences in perfusion recovery which was similar at two weeks in all the strains of mice. TLR2 KO mice demonstrated abnormal vessel morphology compared to other strains and impaired tube formation in vitro.

Discussion

TLR2 and TRIF signaling are necessary for muscle regeneration after ischemia while MyD88 may instead mediate muscle injury. The absence of TLR4 did not affect muscle responses to ischemia. TLR4 may mediate inflammatory responses through MyD88 that are exaggerated in the absence of TLR2. Additionally, the actions of TLR4 through TRIF may promote regenerative responses that are required for recovery from muscle ischemia.