Rac1 and Cdc42 capture microtubules through IQGAP1 and CLIP-170

Linkage of microtubules to special cortical regions is essential for cell polarization. CLIP-170 binds to the growing ends of microtubules and plays pivotal roles in orientation. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts with CLIP-170. In Vero fibroblasts, IQGAP1 localizes at the polarized leading edge. Expression of carboxy-terminal fragment of IQGAP1, which includes the CLIP-170 binding region, delocalizes GFP-CLIP-170 from the tips of microtubules and alters the microtubule array. Activated Rac1/Cdc42, IQGAP1, and CLIP-170 form a tripartite complex. Furthermore, expression of an IQGAP1 mutant defective in Rac1/Cdc42 binding induces multiple leading edges. These results indicate that Rac1/Cdc42 marks special cortical spots where the IQGAP1 and CLIP-170 complex is targeted, leading to a polarized microtubule array and cell polarization.     

Complete Mitochondrial DNA Sequence of Tigriopus japonicus (Crustacea: Copepoda)

The complete nucleotide sequence of the mitochondrial genome was determined for a harpacticoid copepod, Tigriopus japonicus (Crustacea), using an approach that employs a long polymerase chain reaction technique and primer walking. Although the genome (14,628 bp) contained the same set of 37 genes (2 ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes) as found in other metazoan animals, none of the previously reported gene orders were comparable to that of T. japonicus. Furthermore, all genes were encoded on one strand, unlike the mitochondrial genomes of most metazoan animals. Size reductions were notable for tRNA and rRNA genes, resulting in one of the smallest mitochondrial genomes in the arthropod lineage. Although it appears that such large-scale gene rearrangements have occurred in the ancestral species of T. japonicus, none of the proposed mechanisms parsimoniously account for this eccentric gene arrangement.     

Roles of Rho-associated Kinase in Cytokinesis; Mutations in Rho-associated Kinase Phosphorylation Sites Impair Cytokinetic Segregation of Glial Filaments

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, regulates formation of stress fibers and focal adhesions, myosin fiber organization, and neurite retraction through the phosphorylation of cytoskeletal proteins, including myosin light chain, the ERM family proteins (ezrin, radixin, and moesin) and adducin. Rho-kinase was found to phosphorylate a type III intermediate filament (IF) protein, glial fibrillary acidic protein (GFAP), exclusively at the cleavage furrow during cytokinesis. In the present study, we examined the roles of Rho-kinase in cytokinesis, in particular organization of glial filaments during cytokinesis. Expression of the dominant-negative form of Rho-kinase inhibited the cytokinesis of Xenopus embryo and mammalian cells, the result being production of multinuclei. We then constructed a series of mutant GFAPs, where Rho-kinase phosphorylation sites were variously mutated, and expressed them in type III IF-negative cells. The mutations induced impaired segregation of glial filament (GFAP filament) into postmitotic daughter cells. As a result, an unusually long bridge-like cytoplasmic structure formed between the unseparated daughter cells. Alteration of other sites, including the cdc2 kinase phosphorylation site, led to no remarkable defect in glial filament separation. These results suggest that Rho-kinase is essential not only for actomyosin regulation but also for segregation of glial filaments into daughter cells which in turn ensures correct cytokinetic processes.     

Nitrite-Specific Active Transport System of the Cyanobacterium Synechococcus sp. Strain PCC 7942

Studies on the nitrite uptake capability of a mutant of Synechococcus sp. strain PCC 7942 lacking the ATP-binding cassette-type nitrate-nitrite-bispecific transporter revealed the occurrence of a nitrite-specific active transport system with an apparent K_m (NO₂⁻) of about 20 μM. Similar to the nitrate-nitrite-bispecific transporter, the nitrite-specific transporter was reversibly inhibited by ammonium in the medium.     

The induction of murine tumor infiltrating lymphocytes (TIL) by interleukin-2 or T cell growth factor

Mice were injected in the foot pad with either 5×10⁵ syngeneic plasmacytoma (MOPC104E) or fibrosarcoma cells (Meth A). Lymph nodes containing tumor cells were harvested 14 days later and cultured. In the presence of recombinant interleukin-2 (r-IL-2) predominantly tumor cells proliferated. Culture with T cell growth factor (TCGF) resulted in the growth of lymphoid cells. Concanavalin A (Con A) had only a modest effect on elimination of tumor cells in the culture. Tumor-infiltrating lymphocytes (TIL) prepared from the lymph nodes showed specific tumor-neutralizing activity when grown in the presence of TCGF. In vitro examination revealed that Meth A cells could not be lysed by TIL, while TIL from MOPC tumors showed tumor specific activity. This study may explain negative results in human trials with TIL induced by IL-2 alone.Abbreviations r recombinant - IL-2 interleukin-2 - TCGF T cell growth factor - TIL tumor infiltrating lymphocytes - Con A concanavalin A - HBSS Hanks' balanced salt solution     

Sequence-specific 1H and 15N resonance assignments and secondary structure of GDP-bound human c-Ha-Ras protein in solution

Summary All the backbone ¹H and ¹⁵N magnetic resonances (except for Pro residues) of the GDP-bound form of a truncated human c-Ha-ras proto-oncogene product (171 amino acid residues, the Ras protein) were assigned by ¹⁵N-edited two-dimensional NMR experiments on selectively ¹⁵N-labeled Ras proteins in combination with three-dimensional NMR experiments on the uniformly ¹⁵N-labeled protein. The sequence-specific assignments were made on the basis of the nuclear Overhauser effect (NOE) connectivities of amide protons with preceding amide and/or Cprotons. In addition to sequential NOEs, vicinal spin coupling constants for amide protons and C protons and deuterium exchange rates of amide protons were used to characterize the secondary structure of the GDP-bound Ras protein; six strands and five helices were identified and the topology of these elements was determined. The secondary structure of the Ras protein in solution was mainly consistent with that in crystal as determined by X-ray analyses. The deuterium exchange rates of amide protons were examined to elucidate the dynamic properties of the secondary structure elements of the Ras protein in solution. In solution, the -sheet structure in the Ras protein is rigid, while the second helix (A66-R73) is much more flexible, and the first and fifth helices (S17-124 and V152-L171) are more rigid than other helices. Secondary structure elements at or near the ends of the effector-region loop were found to be much more flexible in solution than in the crystalline state.     

MSl3, a multicopy suppressor of mutants hyperactivated in the RAS-CAMP pathway, encodes a novel HSP70 protein of Saccharomyces cerevisiae

The MSI3 gene was isolated as a multicopy suppressor of the heat shock-sensitive phenotype of the iral mutation, which causes hyperactivation of the RAS-cAMP pathway. Overexpression of MSI3 also suppresses the heat shock-sensitive phenotype of the bcyl mutant. Determination of the DNA sequence of MSI3 revealed that MSI3 can encode a 77.4 kDa protein related to the HSP70 family. The amino acid sequence of Msi3p is about 30% identical to that of the Ssalp of Saccharomyces cerevisiae. This contrasts with the finding that members of the HSP70 family generally show at least 50% amino acid identity. The consensus nucleotide sequence of the heat shock element (HSE) was found in the upstream region of MSI3. Moreover, the steady-state levels of the MSI3 mRNA and protein were increased upon heat shock. These results indicate that the MSI3 gene encodes a novel HSP70-like heat shock protein. Disruption of the MSI3 gene was associated with a temperature sensitive growth phenotype but unexpectedly, thermotolerance was enhanced in the disruptant.