K-ras Gene Mutation Related to Histological Atypias in Human Colorectal Adenomas

To investigate the relationship of oncogene analysis to morphology, we analyzed K-ras gene mutations by dot-blot hybridization with and without consideration of histological atypias in individual colorectal adenomas. Each of 54 colon polyps were divided into two parts after fixation. One part was used as a mass to assess point mutations; the remaining portion of each polyp was paraffin-embedded, stained with hematoxylin and eosin, and examined for point mutations related to histological atypias. In the first part of our study, K-ras gene mutations at codon 12 were detected in 13 cases (24%). In the second part of our study, 12 cases had distinctly different histological atypias. From each of these 12 cases, two areas, one with higher or one with lower grade atypia in the same polyp were excised to analyze for K-ras gene mutation. Two of these 12 cases (17%) had the mutation in different areas of the same tumor. These two cases contained the mutation only in the areas with higher grade atypia, and only one case added information regarding ras mutation upon microdissection when compared to the entire biopsy. These results suggest that oligonucleotide hybridization can identify the majority of cases containing ras mutations despite regional morphologic variation. Individual cases, however, may contain clonal subpopulations within adenomas with different ras sequences from other regions within the same adenoma.     

Proliferative response of seminal vesicle cells to androgen and estrogen in neonatally castrated mice

Proliferation and death of androgen- and estrogen-responsive cells in seminal vesicles were compared between neonatally and adult (on Day 60 after birth) castrated mice. Daily injections of either testosterone propionate (TP) or estradiol-17 beta (E2) were started on Day 90 after birth; the incorporation of 5-[125I]iodo-2'-deoxyuridine ([125I]IdUrd) into the whole seminal vesicles was used as an index for proliferation. Although the peak of [125I]IdUrd uptake was observed 3 days after starting TP injections in both neonatally and adult castrated mice, the peak was lower and the period of proliferation was much longer in the former than in the latter. When TP injections were stopped, the fraction of surviving cells that synthesized DNA on Day 3 of TP injections was much larger in neonatally than adult castrated mice. The difference was attributed to the presence of TP-induced proliferation of fibromuscular cells in the neonatally castrated mice but not in the adult castrated mice; only the fibromuscular cells but not epithelial cells survived after stopping TP injections. Although injections of E2 increased the proliferation of epithelial cells but did not the weight of seminal vesicles in adult castrated mice, the same procedure increased the proliferation of both epithelial and fibromuscular cells and the weight in neonatally castrated mice. The E2-induced fibromuscular cells seemed to survive in the presence or absence of E2. The present results seem to indicate that androgen- and estrogen-induced proliferation of fibromuscular cells is irreversible in seminal vesicles of neonatally castrated mice and that the depletion of androgen in the seminal vesicle during neonatal and prepubertal periods is at least in part compensated by the administration of androgen, even after 90 days of age.     

Repression of porin synthesis by salicylate in Escherichia coli, Klebsiella pneumoniae and Serratia marcescens

The synthesis of the OmpF porin in Escherichia coli K-12 was highly and reversibly inhibited by 5 mM salicylate in the bacterial growth medium, and salicylate also inhibited the OmpC porin synthesis, although only weakly. The full expression of the salicylate effect was presumed to require the ompB gene product on comparison between the wild type and ompB mutant strains. The salicylate effect was also observed for the porin protein synthesis in Klebsiella pneumoniae and Serratia marcescens, although an ompB-like gene remains to be identified in both species.     

Bacterial expression and characterization of molluscan IDO-like myoglobin

The indoleamine 2,3-dioxygenase (IDO)-like myoglobin (Mb) is a unique type of Mb isolated from the buccal mass of several archgastropod species. Here, we expressed Sulculus diversicolor IDO-like Mb as a GST-fusion protein in bacteria. The visible spectrum of GST-fusion IDO-like Mb shows characteristic α- and β-peaks, indicating that it binds oxygen. To identify residues important in heme and oxygen binding, we constructed site-directed mutants. We initially replaced each of the 7 histidines of S. diversicolor IDO-like Mb with alanine. The spectra of three mutants (H74A, H288A, and H332A) revealed a remarkable loss of absorbance around 414 nm, indicating that they cannot bind heme. His⁷⁴, His²⁸⁸, and His³³² were also replaced by arginine or tyrosine. Neither H332R nor H332Y contains heme, suggesting that His³³² is the proximal ligand of IDO-like Mb. In contrast, both H74R and H288Y mutants were isolated in the heme-binding oxy-form. The autoxidation rates of these two mutants showed that they can bind oxygen as stably as wild-type. His⁷⁴ and His²⁸⁸ might be partially associated with heme-binding, but do not act as the distal ligand. The S. diversicolor IDO-like Mb seems to stably bind oxygen in a different manner from normal myoglobins.     

The extracellular domain of p185(c-neu) induces density-dependent inhibition of cell growth in malignant mesothelioma cells and reduces growth of mesothelioma in vivo

EGFR is involved in the density-dependent inhibition of cell growth, while coexpression of EGFR with erbB2 can render normal cells transformed. In this study, we have examined the effect of a species of p185 that contains the transmembrane domain and the extracellular domain of p185(c-neu), on growth properties of a human malignant mesothelioma cell line that coexpresses EGFR and erbB2. The ectodomain form of p185(c-neu) enhanced density-dependent inhibition of cell growth and we found that p21 induction appeared to be responsible for this inhibitory effect. Previously, the extracellular domain species was shown to suppress the transforming abilities of EGFR and p185(c-neu/erbB2) in a dominant-negative manner. The ability of this subdomain to affect tumor growth is significant, as it reduced in vivo tumor growth. Unexpectedly, we found that the domain did not abrogate all of EGFR functions. We noted that EGFR-induced density-dependent inhibition of cell growth was retained. Tyrosine kinase inhibitors of EGFR did not cause density-dependent inhibition of cell growth of malignant mesothelioma cells. Therefore, simultaneously inhibiting the malignant phenotype and inducing density-dependent inhibition of cell growth in malignant mesothelioma cells by the extracellular domain of p185(c-neu) may represent an important therapeutic advance.     

Efficient substructure RMSD query algorithms.

Protein structure analysis is a very important research topic in the molecular biology of the post-genomic era. The root mean square deviation (RMSD) is the most frequently used measure for comparing two protein three-dimensional (3-D) structures. In this paper, we deal with two fundamental problems related to the RMSD. We first deal with a problem called the "range RMSD query" problem. Given an aligned pair of structures, the problem is to compute the RMSD between two aligned substructures of them without gaps. This problem has many applications in protein structure analysis. We propose a linear-time preprocessing algorithm that enables constant-time RMSD computation. Next, we consider a problem called the "substructure RMSD query" problem, which is a generalization of the above range RMSD query problem. It is a problem to compute the RMSD between any substructures of two unaligned structures without gaps. Based on the algorithm for the range RMSD problem, we propose an O(nm) preprocessing algorithm that enables constant-time RMSD computation, where n and m are the lengths of the given structures. Moreover, we propose O(nm log r/r)-time and O(nm/r)-space preprocessing algorithm that enables O(r) query, where r is an arbitrary integer such that 1 < or = r < or = min(n, m). We also show that our strategy also works for another measure called the unit-vector root mean square deviation (URMSD), which is a variant of the RMSD.     

Quantitative analysis of changes in cell shape of Amoeba proteus during locomotion and upon responses to salt stimuli

A new parameter expressing the complexity of cell shape defined as (periphery)²/(area) in 2D projection was found useful for a quantitative analysis of changes in the cell shape of Amoeba proteus and potentially of any amoeboid cells. During locomotion the complexity and the motive force of the protoplasmic streaming in amoeba varied periodically, and the Fourier analysis of the two showed a similar pattern in the power spectrum, giving a rather broad peak at about 2.5 × 10⁻³ Hz. The complexity increased mainly due to elongation of the cell as external Ca²⁺ increased. This effect was blocked by La³⁺, half the inhibition being attained at 1/200 amount of the coexisting Ca²⁺. On the other hand, the complexity decreased due to rounding up of the cell as the concentration of other cations, such as Sr²⁺, Mg²⁺, Co²⁺, Ni²⁺, Na⁺, K⁺ etc., increased. Irrespective of the opposite effects of Ca²⁺ and other cations on the cell shape, the ATP concentration in amoeba decreased in both cases with increase of all these cations. The irregularity in amoeboid motility is discussed in terms of a dynamic system theory.