ROOT GROWTH INHIBITING, a Rice Endo-1,4-β-d-Glucanase, Regulates Cell Wall Loosening and is Essential for Root Elongation

The molecular mechanism involved in cell wall dynamics has not been well clarified, although it is quite important for organ growth. We characterized a rice mutant, root growth inhibiting (rt), which is defective in root elongation. The rt mutant showed a severe defect in cell elongation at the root-elongating zone with additional collapse of epidermal and cortex cells at the root tip caused by the defect in the smooth exfoliation of root cap cells. Consistent with these phenotypes, expression of the RT gene, which encodes a member of the membrane-anchored endo-1,4-??-d-glucanase, was specifically localized in the root-elongating zone and at the junction between epidermal and root cap cells. The enzymatic analysis of root extracts from the wild-type and rt mutant indicated that RT hydrolyzes noncrystalline amorphous cellulose. The cellulose content was slightly increased but the crystallinity of cellulose was decreased in the rt root. In addition, the hemicellulose composition was different between wild-type and rt roots. The total extensibility was significantly lower in the rt root explants. Based on these results, we concluded that RT is involved in the disassembly of the cell wall for cell elongation in roots as well as for root cap exfoliation from the epidermal cell layer by hydrolyzing the noncrystalline amorphous cellulose fibers of cellulose microfibrils resulting in loosening of the hemicellulose and cellulose interaction.     

Uterine epithelial cells specifically induce interferon-stimulated genes in response to polyinosinic-polycytidylic acid independently of estradiol

Interferon β (IFNβ) is an antiviral cytokine secreted in response to pathogenic exposure that creates a restrictive intracellular environment through the action of downstream interferon-stimulated genes (ISG). The objective of this study was to examine the expression of IFNβ and ISG in both human uterine epithelial cells (UEC) and the ECC-1 uterine epithelial cell line and determine if expression changes with TLR stimulation and hormone exposure. Stimulation of primary uterine epithelial cells and ECC-1 cells with the TLR3 agonist poly (I:C) induced the mRNA expression of IFNβ, MxA, OAS2 and PKR. Other TLR agonists including imiquimod and CpG had no effect on either IFNβ or ISG expression. In contrast to ECC-1 cell responses which were slower, maximal IFNβ upregulation in UEC occurred 3 hours post-stimulation and preceded the ISG response which peaked approximately 12 hours after poly (I:C) exposure. Unexpectedly, estradiol, either alone or prior to treatment with poly (I:C), had no effect on IFNβ or ISG expression. Blockade of the IFN receptor abrogated the upregulation of MxA, OAS2 and PKR. Furthermore, neutralizing antibodies against IFNβ partially inhibited the upregulation of all three ISG. Estradiol, directly and in the presence of poly (I:C) had no effect on IFNβ and ISG expression. These results indicate that uterine epithelial cells are important sentinels of the innate immune system and demonstrate that uterine epithelial cells are capable of mounting a rapid IFN-mediated antiviral response that is independent of estradiol and is therefore potentially sustained throughout the menstrual cycle to aid in the defense of the uterus against potential pathogens.     

YIPF5 and YIF1A recycle between the ER and the Golgi apparatus and are involved in the maintenance of the Golgi structure

Yip1p/Yif1p family proteins are five-span transmembrane proteins localized in the Golgi apparatus and the ER. There are nine family members in humans, and YIPF5 and YIF1A are the human orthologs of budding yeast Yip1p and Yif1p, respectively. We raised antisera against YIPF5 and YIF1A and examined the localization of endogenous proteins in HeLa cells. Immunofluorescence, immunoelectron microscopy and subcellular fractionation analysis suggested that YIPF5 and YIF1A are not restricted to ER exit sites but also localized in the ER-Golgi intermediate compartment (ERGIC) and some in the cis-Golgi at steady state. Along with ERGIC53, YIPF5 and YIF1A remained in the cytoplasmic punctate structures after brefeldin A treatment, accumulated in the ERGIC and the cis-Golgi after treatment with AlF₄^- and accumulated in the ER when ER to Golgi transport was inhibited by Sar1(H79G). These results supported the localization of YIPF5 and YIF1A in the ERGIC and the cis-Golgi, and strongly suggested that they are recycling between the ER and the Golgi apparatus. Analysis by blue native PAGE and co-immunoprecipitation showed that YIPF5 and YIF1A form stable complexes of three different sizes. Interestingly, the knockdown of YIPF5 or YIF1A caused partial disassembly of the Golgi apparatus suggesting that YIPF5 and YIF1A are involved in the maintenance of the Golgi structure.     

Coupling of DNA binding and helicase activity is mediated by a conserved loop in the MCM protein

Minichromosome maintenance (MCM) helicases are the presumptive replicative helicases, thought to separate the two strands of chromosomal DNA during replication. In archaea, the catalytic activity resides within the C-terminal region of the MCM protein. In Methanothermobacter thermautotrophicus the N-terminal portion of the protein was shown to be involved in protein multimerization and binding to single and double stranded DNA. MCM homologues from many archaeal species have highly conserved predicted amino acid similarity in a loop located between β7 and β8 in the N-terminal part of the molecule. This high degree of conservation suggests a functional role for the loop. Mutational analysis and biochemical characterization of the conserved residues suggest that the loop participates in communication between the N-terminal portion of the helicase and the C-terminal catalytic domain. Since similar residues are also conserved in the eukaryotic MCM proteins, the data presented here suggest a similar coupling between the N-terminal and catalytic domain of the eukaryotic enzyme.     

Probes for Catalytic Action of α-Chymotrypsin in Plastein Synthesis

A plastein was synthesized with α-chymotrypsin from a dialyzable fraction of a peptic hydrolysate of soybean protein.

The plastein was obtainable also by use of an insoluble preparation of α-chymotrypsin. This may rule out the possibility that the plastein is a product resulting from some chemical peptide-protein (enzyme) aggregation.

No appreciable amount of the plastein was produced when chymotrypsinogen was used instead of α-chymotrypsin.

The plastein synthetic, as well as the protein hydrolytic, activity of α-chymotrypsin was inhibited more or less by a hydrophobic inhibitor (n-hexane), a competitive inhibitor (benzolyl-d,l-phenylalanine), and divalent cations (Zn²⁺, Hg²⁺ and Cu²⁺); the degree of inhibition in each case was approximately similar against both the synthetic and the hydrolytic activities.

Either diisopropylphosphorylation of the β-O of Ser-195 or methylation of the 3-N of His-57 imidazole of α-chymotrypsin repressed the synthetic, as well as the hydrolytic, activity.

Based on these results a possible mechanism was discussed of the plastein synthesis by α-chymotrypsin, especially in relevance to its acylation and deacylation.     

Survival of Escherichia coli under lethal heat stress by L-form conversion

Transition of bacteria to cell wall deficient L-forms in response to stress factors has been assumed as a potential mechanism for survival of microbes under unfavorable conditions. In this article, we provide evidence of paradoxal survival through L-form conversion of E. coli high cell density population after lethal treatments (boiling or autoclaving). Light and transmission electron microscopy demonstrated conversion from classical rod to polymorphic L-form shape morphology and atypical growths of E. coli. Microcrystal formations observed at this stage were interpreted as being closely linked to the processes of L-form conversion and probably involved in the general phenomenon of protection against lethal environment. Identity of the morphologically modified L-forms as E. coli was verified by species specific DNA-based test. Our study might contribute to a better understanding of the L-form phenomenon and its importance for bacterial survival, as well as provoke reexamination of the traditional view of killing strategies against bacteria.     

The Virulence Plasmid of Salmonella typhimurium Is Self-Transmissible

Most isolates of Salmonella enterica serovar Typhimurium contain a 90-kb virulence plasmid. This plasmid is reported to be mobilizable but nonconjugative. However, we have determined that the virulence plasmid of strains LT2, 14028, and SR-11 is indeed self-transmissible. The plasmid of strain SL1344 is not. Optimal conjugation frequency requires filter matings on M9 minimal glucose plates with a recipient strain lacking the virulence plasmid. These conditions result in a frequency of 2.9 × 10⁻⁴ transconjugants/donor. Matings on Luria-Bertani plates, liquid matings, or matings with a recipient strain carrying the virulence plasmid reduce the efficiency by up to 400-fold. Homologs of the F plasmid conjugation genes are physically located on the virulence plasmid and are required for the conjugative phenotype.