The amino acid sequences of two alpha chains of hemoglobins from Komodo dragon Varanus komodoensis and phylogenetic relationships of amniotes

To elucidate phylogenetic relationships among amniotes and the evolution of alpha globins, hemoglobins were analyzed from the Komodo dragon (Komodo monitor lizard) Varanus komodoensis, the world's largest extant lizard, inhabiting Komodo Islands, Indonesia. Four unique globin chains (alpha A, alpha D, beta B, and beta C) were isolated in an equal molar ratio by high performance liquid chromatography from the hemolysate. The amino acid sequences of two alpha chains were determined. The alpha D chain has a glutamine at E7 as does an alpha chain of a snake, Liophis miliaris, but the alpha A chain has a histidine at E7 like the majority of hemoglobins. Phylogenetic analyses of 19 globins including two alpha chains of Komodo dragon and ones from representative amniotes showed the following results: (1) The a chains of squamates (snakes and lizards), which have a glutamine at E7, are clustered with the embryonic alpha globin family, which typically includes the alpha D chain from birds; (2) birds form a sister group with other reptiles but not with mammals; (3) the genes for embryonic and adult types of alpha globins were possibly produced by duplication of the ancestral alpha gene before ancestral amniotes diverged, indicating that each of the present amniotes might carry descendants of the two types of alpha globin genes; (4) squamates first split off from the ancestor of other reptiles and birds.      

A novel mammalian Ras GTPase-activating protein which has phospholipid-binding and Btk homology regions.

We have previously purified a novel GTPase-activating protein (GAP) for Ras which is immunologically distinct from the known Ras GAPs, p120GAP and neurofibromin (M. Maekawa, S. Nakamura, and S. Hattori, J. Biol. Chem. 268:22948-22952, 1993). On the basis of the partial amino acid sequence, we have obtained a cDNA which encodes the novel Ras GAP. The predicted protein consists of 847 amino acids whose calculated molecular mass, 96,369 Da, is close to the apparent molecular mass of the novel Ras GAP, 100 kDa. The amino acid sequence shows a high degree of similarity to the entire sequence of the Drosophila melanogaster Gap1 gene. When the catalytic domain of the novel GAP was compared with that of Drosophila Gap1, p120GAP, and neurofibromin, the highest degree of similarity was again observed with Gap1. Thus, we designated this gene Gap1m, a mammalian counterpart of the Drosophila Gap1 gene. Expression of Gap1m was relatively high in brain, placenta, and kidney tissues, and it was expressed at low levels in other tissues. A recombinant protein consisting of glutathione-S-transferase and the GAP-related domain of Gap1m stimulated GTPase of normal Ras but not that of Ras having valine at the 12th residue. Expression of the same region in Saccharomyces cerevisiae suppressed the ira2- phenotype. In addition to the GAP catalytic domain, Gap1m has two domains with sequence closely related to those of the phospholipid-binding domain of synaptotagmin and a region with similarity to the unique domain of Btk tyrosine kinase. These results clearly show that Gap1m is a novel Ras GAP molecule of mammalian cells.     

Effect of shock-loading of heavy metals on total organic carbon and phosphate removal in an anaerobic-aerobic activated sludge process

The effect of shock-loading of zinc, copper and cadmium ions on the removal of total organic carbon (TOC) and phosphate in an anaerobic-aerobic activated sludge process was investigated. TOC removal was not sensitive to shock-loading of Zn²⁺ and Cd²⁺ ions, and complete removal was achieved even at 20 mg Zn²⁺/l and 20 mg Cd²⁺/l. However, with over 1 mg Cu²⁺/1 TOC removal efficiency decreased. PO _inf4 ^sup3- removal, in contrast, was extremely sensitive to these metal ions, with the threshold being 1 mg Zn²⁺/l and 1 mg Cd²⁺/l. Higher concentrations adversely affected PO _inf4 ^sup3- removal. Copper again proved detrimental; no PO _inf4 ^sup3- removal was achieved even at 1 mg Cu/l. These results highlight the sensitivity of the removal efficiencies of TOC and PO _inf4 ^sup3- to shock loadings of these heavy metals.Y.P. Ting is with the Department of Chemical Engineering, National University of Singapore, Kent Ridge, 0511, Singapore; H. Imai and S. Kinoshita are with the Department of Chemical Process Engineering, Hokkaido University, Sapporo 060, Japan.     

Construction of a Biotin-Overproducing Strain of Serratia marcescens

We have isolated mutants resistant to acidomycin, a biotin analog, from Serratia marcescens Sr41. Strain SB304, resistant to 0.5 mg of acidomycin (frequently called actithiazic acid) per ml, produced 5 mg of d-biotin per liter of a medium containing sucrose and urea. Strain SB412, which was isolated from SB304 on a minimal agar plate containing 2 mg of acidomycin per ml and 0.1 mg of 5-(2-thienyl)-valeric acid per ml, produced 20 mg of d-biotin per ml. The two enzymes related to biotin synthesis were found to be released from biotin-mediated feedback repression in these mutants. Transductional analysis revealed that SB412 had acquired at least two mutations, one in the biotin operon locus and the other in an unknown locus distant from the biotin operon locus.     

Characterization of a temperature-sensitive influenza B virus mutant defective in neuraminidase.

ts5, a temperature-sensitive mutant of influenza B virus, belongs to one of seven recombination groups. When the mutant infected MDCK cells at the nonpermissive temperature (37.5 degrees C), infectious virus was produced at very low levels compared with the yield at the permissive temperature (32 degrees C) and hemagglutinating and enzymatic activities were undetectable. However, viral protein synthesis and transport of hemagglutinin (HA) and neuraminidase (NA) to the cell surface were not affected. The NA was found as a monomer within cells even at 32 degrees C, in contrast to wild-type virus NA, existing mostly as an oligomer, but the mutant had oligomeric NA, like the wild-type virus. Its enzymatic activity was more thermolabile than that of wild-type virus. Despite the low yield, large aggregates of progeny virus particles were found to accumulate on the cell surface at the nonpermissive temperature, and these aggregates were broken by treatment with bacterial neuraminidase, with the concomitant appearance of hemagglutinating activity, suggesting that NA prevents the aggregation of progeny virus by removal of neuraminic acid from HA and cell receptor, allowing its release from the cells. Further treatment with trypsin resulted in the recovery of infectivity. When bacterial NA was added to the culture early in infection, many hemagglutinable infectious virus was produced. We also suggest that the removal of neuraminic acid from HA by NA is essential for the subsequent cleavage of HA by cellular protease. Nucleotide sequence analysis of RNA segment 6 revealed that ts5 encoded five amino acid changes in the NA molecule but not in NB.     

Molecular breeding of a biotin-hyperproducing Serratia marcescens strain.

We previously reported that an acidomycin-resistant mutant of Serratia marcescens Sr41, SB304, and a mutant that was derived from SB304 and was resistant to a higher concentration of acidomycin, SB412, produced 5 and 20 mg of D-biotin, respectively, per liter of a medium containing sucrose and urea (N. Sakurai, Y. Imai, M. Masuda, S. Komatsubara, and T. Tosa, Appl. Environ. Microbiol. 59:2857-2863, 1993). In order to increase the productivity of D-biotin, the biotin (bio) operons were cloned from strains SB412, SB304, and 8000 (wild-type strain), and pLGM412, pLGM304, and pLGW101, respectively, were obtained through subcloning. These plasmids harbored 7.2-kb DNA fragments coding for the bioABFCD genes on a low-copy-number vector and were introduced into SB304, SB412, and 8000. Among the resulting recombinant strains, SB412(pLGM304) exhibited the highest D-biotin production (200 mg/liter) in the production medium. The plasmid was stably maintained in cells. Unexpectedly, SB412(pLGM412) grew very slowly, and the D-biotin productivity of this recombinant strain was not evaluated because pLGM412 was unstable.     

Ischemic changes in myocardial ionic contents of the isolated perfused rat hearts as studied by NMR

Using³¹P-,²³Na- and³⁹K-NMR, we assessed ischemic changes in high energy phosphates and ion contents of isolated perfused rat hearts continuously and systematically. To discriminate intra- and extracellular Na⁺, a shift reagent (Dy(TTHA)^3–) was used in²³Na-NMR study. In³⁹K-NMR study, the extracellular K⁺ signal was suppressed by inversion recovery pulse sequence in order to obtain intracellular K⁺ signal without using shift reagnets. During the early period of ischemia, increases in intracellular Na⁺ and inorganic phosphate (Pi) were observed in addition to the well-documented decreases in creatine phosphate and ATP and a fall of intracellular pH, suggesting an augmented operation of Na⁺–H⁺ exchange triggered by a fall of the intracellular pH resulted from breakdown of ATP. At around 15 min of ischemia, a second larger increase in intracellular Na⁺ and a decrease in intracellular K⁺ were observed in association with a second increase in Pi. This was accompnanied by an abrupt rise of the ventricular end-diastolic pressure. As there was a depletion of ATP at this time, the increase in intracellular Na⁺ and associated decrease in intracellular K⁺ may be explained by inhibition of the Na⁺–K⁺ ATPase due to the depletion of ATP. A longer observation with³¹P-NMR revealed a second phosphate peak (at lower magnetic field to ordinary Pi peak) which increased its intensity as ischemic time lengthened. The pH of this 2nd peak changed in parallel with the changes in pH of the bathing solution, indicating the appearance of a compartment whose hydrogen concentration is in equilibrium with that of the external compartment. Thus, the peak could be used as an index of irreversible membrane damage of the myocardium.     

Effects of body and head positions on bilateral difference in tympanic temperatures

We have examined the nonparallel changes in tampanic membrane temperatures (T _ty) from the two ears in response to various changes in body and head positions. Upon assuming a lateral recumbent position, the T _ty on the lower side increased while that on the upper side decreased. Pressure application over a wide area of the lateral chest only caused inconsistent and obscure asymmetric changes in T _ty. A lateral flexion of the head with the subject sitting upright and a rotation of the head to the side in a supine position induced an increase in the T _ty on the lower side compared to that on the upper side. The temperature and blood flow of the forehead often decreased on the lower side and increased on the upper side, although such responses were not always concomitant with the asymmetric changes in T _ty. A dorsal flexion of the head with the subject in a reclining position caused a slight increase in the T _ty, whereas raising the head upright induced a slight decrease in them. Two additional experiments were carried out with single photon emission computed tomography using ^99mTc-hexamethylpropyleneamine oxime as tracer, and a slight, relative decrease in counts was noted in the right hemisphere during rotation of the head to the right. These results would strongly suggest that unilateral increases and decreases in T _ty could have been caused by one-sided decreases and increases, respectively, in blood flow to the brain and/or the tympanic membrane, induced by a vasomotor reflex involving vestibular stimulation.