Functional and structural conservation in the mechanosensitive channel MscL implicates elements crucial for mechanosensation

mscL encodes a channel in Escherichia coli that is opened by membrane stretch force, probably serving as an osmotic gauge. Sequences more or less similar to mscL are found in other bacteria, but the degree of conserved function has been unclear. We subcloned and expressed these putative homologues in E . coli and examined their products under patch clamp. Here, we show that each indeed encodes a conserved mechanosensitive channel activity, consistent with the interpretation that this is an important and primary function of the protein in a wide range of bacteria. Although similar, channels of different bacteria differ in kinetics and their degree of mechanosensitivity. Comparison of the primary sequence of these proteins reveals two highly conserved regions, corresponding to domains previously shown to be important for the function of the wild-type E . coli channel, and a C-terminal region that is not conserved in all species. This structural conservation is providing insight into regions of this molecule that are vital to its role as a mechanosensitive channel and may have broader implications for the understanding of other mechanosensitive systems.     

IPA-3 Inhibits the Growth of Liver Cancer Cells By Suppressing PAK1 and NF-κB Activation

Hepatocellular carcinoma (HCC) is one of the major malignancies worldwide and is associated with poor prognosis due to the high incidences of metastasis and tumor recurrence. Our previous study showed that overexpression of p21-activated protein kinase 1 (PAK1) is frequently observed in HCC and is associated with a more aggressive tumor behavior, suggesting that PAK1 is a potential therapeutic target in HCC. In the current study, an allosteric small molecule PAK1 inhibitor, IPA-3, was evaluated for the potential in suppressing hepatocarcinogenesis. Consistent with other reports, inhibition of PAK1 activity was observed in several human HCC cell lines treated with various dosages of IPA-3. Using cell proliferation, colony formation and BrdU incorporation assays, we demonstrated that IPA-3 treatment significantly inhibited the growth of HCC cells. The mechanisms through which IPA-3 treatment suppresses HCC cell growth are enhancement of apoptosis and blockage of activation of NF-κB. Furthermore, our data suggested that IPA-3 not only inhibits the HCC cell growth, but also suppresses the metastatic potential of HCC cells. Nude mouse xenograft assay demonstrated that IPA-3 treatment significantly reduced the tumor growth rate and decreased tumor volume, indicating that IPA-3 can suppress the in vivo tumor growth of HCC cells. Taken together, our demonstration of the potential preclinical efficacy of IPA-3 in HCC provides the rationale for cancer therapy.     

Evolution of the thaumastocheliform lobsters (Crustacea,Decapoda, Nephropidae)

Deep‐sea lobsters previously assigned to the family Thaumastochelidae Bate, 1888, the thaumastocheliforms, have very distinctive, greatly unequal first chelipeds, with the right side extremely elongate and pectinate, and in having short, quadrate pleonal pleura. Despite interesting morphology and a long taxonomic history, the phylogeny of the group has received little detailed analysis. Here, we conduct a species‐level phylogenetic analysis of the thaumastocheliforms based on morphological and molecular data (three mitochondrial genes: COI, 16S rDNA and 12S rDNA; two nuclear protein‐coding genes: H3 and NaK) to robustly reconstruct their evolutionary history and estimate divergence times. Separate and combined analyses of all data sources support thaumastocheliform monophyly, but as a clade deeply nested within the Nephropidae supporting recent synonymy of Thaumastochelidae with Nephropidae. Combined and molecular‐only analyses support generic monophyly of all three thaumastocheliform genera and Dinochelus as sister to Thaumastochelopsis, fully corroborating the current, morphology‐based taxonomy. In contrast, Thaumastocheles is recovered as paraphyletic in morphology‐only analyses owing to minimal character support. The Cretaceous–Paleogene Oncopareia was recovered as a stem‐lineage thaumastocheliform. The fossil record indicates that the thaumastocheliforms once lived in shallow, continental shelf depths, but moved into deeper water in the Cenozoic where they occur today. The thaumastocheliforms originated in northern Europe during the Mid‐Late Cretaceous and later dispersed westwards to the south‐eastern Pacific through the western Atlantic and eastwards to the western Pacific through the Indian Ocean. Thaumastochelopsis can be considered the most derived thaumastocheliform genus based on the degree of structural reduction relative to other thaumastocheliforms, its remote geographical occurrence (Australia) from the hypothesised place of origin (northern Europe) and its more recent estimated divergence than other genera (28 Mya for the MRCA of extant species of the genus).     

Hypotonic shocks activate rat TRPV4 in yeast in the absence of polyunsaturated fatty acids

Transient-receptor-potential channels (TRPs) underlie the sensing of chemicals, heat, and mechanical force. We expressed the rat TRPV1 and TRPV4 subtypes in yeast and monitored their activities in vivo as Ca²⁺ rise using transgenic aequorin. Heat and capsaicin activate TRPV1 but not TRPV4 in yeast. Hypotonic shocks activate TRPV4 but not TRPV1. Osmotic swelling is modeled to activate enzyme(s), producing polyunsaturated fatty acids (PUFAs) to open TRPV4 in mammalian cells. This model relegates mechanosensitivity to the enzyme and not the channel. Yeast has only a single Δ9 fatty-acid monodesaturase and cannot make PUFAs suggesting an alternative mechanism for TRPV4 activation. We discuss possible explanations of this difference.     

Senescence of rice leaves XXXI. Changes of chlorophyll,protein, and polyamine contents and ethylene production during senescence of a chlorophyll-deficient mutant

Leaf senescence of a chlorophylldeficient rice mutant (LT-8) was investigated. At 10 days after planting, the chlorophyll level in the third leaves of rice seedlings of the mutant was about one half that of normal leaves (Norin no. 8), whereas no difference in the protein level could be detected in the two genotypes. The protein level in leaves decreased with increasing age, and no significant difference could be detected during senescence in the two genotypes. Chlorophyll level in the normal leaves also decreased with increasing age. However, the chlorophyll level in the mutant leaves began to decrease only after more than 60% of the initial protein had been degraded. The pattern of ethylene production in the normal leaves was, in general, similar to that in the mutant leaves. Ethylene production first decreased with age, increased to a maximum at day 18, and decreased thereafter. Both spermidine and spermine levels in the leaves of the two genotypes decreased with increasing age. The pattern of the putrescine level in the normal leaves behaved somewhat similar to that in the mutant leaves. However, during the course of senescence, the putrescine level in the mutant leaves was always higher than that in the normal leaves. The possible relationship between endogenous polyamine levels and ethylene production is discussed.     

Improved Large-Scale Purification of Transducin,and Its α and βγ Subunits from Frozen Retinas

Abstract

The transducin heterotrimer and its α- and βγ-subunits have been purified from frozen bovine rod outer segments by modifying existing procedures. The methods described here are relatively simple and fast. The yield (ca. 8 mgs/100 retinas) and purity of the transducin heterotrimer and subunits from frozen retinas is equal to or larger than those previously obtained from fresh or frozen retinas.