Cloning and sequence analysis of cDNAs encoding mammalian mitochondrial malate dehydrogenase

A cDNA clone, named ppmMDH-1 and covering a part of the porcine mitochondrial malate dehydrogenase (mMDH; L-malate:NAD+ oxidoreductase, EC 1.1.1.37) mRNA, was isolated from a porcine liver cDNA library with a mixture of 24 oligodeoxyribonucleotides as a probe. The sequences of the probe were deduced from the known sequence of porcine mMDH amino acid residues 288-293. ppmMDH-1 covered the coding region for porcine mMDH amino acid residues 17-314 and the 3' untranslated region. Subsequently, mouse mMDH cDNA clones were isolated from a mouse liver cDNA library with the ppmMDH-1 cDNA as a probe. One of the clones, named pmmMDH-1 and containing a cDNA insert of about 1350 base pairs, was selected for sequence analysis, and the primary structure of the mouse precursor form of mMDH (pre-mMDH) was deduced from its cDNA sequence. The sequenced coding regions for the porcine and mouse mMDH mRNAs showed about 85% homology. When the deduced amino acid sequence of the mouse pre-mMDH was compared with that of the porcine mMDH, they shared a 95% homology, and the mouse pre-mMDH yielded a leader sequence consisting of 24 amino acid residues and a mature mMDH, consisting of 314 amino acid residues. The leader sequence contained three basic amino acid residues, no acidic residues, and no hydrophobic amino acid stretch. The mouse mMDH leader sequence was compared with those of three other rodent mitochondrial matrix proteins.     

Inactivation of cytosolic aspartate aminotransferase accompanying modification of Trp 48 by N-bromosuccinimide

Reaction of N-bromosuccinimide with pig heart cytosolic aspartate aminotransferase led to loss of the enzymatic activity. Chemical analysis indicated the modification of two tryptophan residues. At a low ratio of N-bromosuccinimide to enzyme, oxidation of Trp 122 occurred without affecting the enzymatic activity. Increase in the ratio resulted in the oxidation of Trp 48 with a concomitant decrease in enzyme activity. The modified enzyme did not react with substrates and their analogs. Trp 48 is not within the active site but in the hinge region linking the large domain of the enzyme to the small domain that shows dynamic movement upon binding substrates. The present result suggests that oxidation of Trp 48 may impair the structural integrity of the interdomain interface.     

Purification and properties of a pyridoxal 5'-phosphate-dependent histidine decarboxylase from Morganella morganii AM-15

A pyridoxal 5'-phosphate-dependent histidine decarboxylase from Morganella morganii AM-15 was purified to homogeneity. The enzyme is a tetramer (Mr 170,000) of identical subunits and binds 4 pyridoxal-P/tetramer; it is resolved by dialysis against cysteine at pH 6.8. Between pH 6.2 and 8.8, the holoenzyme shows pH-independent absorbance maxima at 333 and 416 nm. Vmax/Km is highest at pH 6.5; this optimum reflects chiefly increased Km values for histidine at lower or higher pH values, whereas Vmax is highest at pH 5.0 and decreases only moderately between pH 5.0 and 8.0. The enzyme also decarboxylates beta-(2-pyridyl)alanine and N tau-methylhistidine (but not N pi-methylhistidine); arginine, lysine, and ornithine are neither substrates nor inhibitors. The hydrazine analogue of histidine, 2-hydrazino-3-(4-imidazolyl)propionic acid, is a very potent competitive inhibitor; other carbonyl reagents and a variety of carboxyl- or amino-substituted histidines also inhibit competitively. alpha-Fluoromethylhistidine is a potent irreversible inhibitor of the enzyme; alpha-methylhistidine is a competitive inhibitor/substrate that is decarboxylated slowly and undergoes a slow decarboxylation-dependent transamination that converts the holoenzyme to pyridoxamine-P and apoenzyme. Dithiothreitol and other simple thiols are mixed-type inhibitors that interact with pyridoxal-P at the active site to form complexes (lambda max congruent to 340 nm), presumably the corresponding thioalkylamines, without resolving the holoenzyme. This histidine decarboxylase (Vmax = 72 mumol X min-1 X mg-1) is much more active than "homogeneous" preparations of mammalian pyridoxal-P-dependent histidine decarboxylase (Vmax congruent to 1.0) and is about equal in activity to the pyruvoyl-dependent histidine decarboxylases from Gram-positive bacteria.     

4-way bred rats as experimental animals

The present study is an attempt to utilize hybrids among several inbred strains of rats as useful animals for the studies of effectiveness and toxicology on drugs., Four-way crosses were made among the LEW, WM, F344 and DRY strains of rats, and their characteristics were examined. From the breeding data of diallel crosses among these four strains and reciprocal crosses among their F1 hybrids, the mating type indicating the highest reproductivity was (LEW X WM) F1 X (F344 X DRY) F1. These four-way crosses were designated as LWFD. The reproductivity of this mating type was exceedingly higher than those of four strains. In order to examine the susceptibility to thiamine hydrochloride, the acute toxicity test was practiced in inbred strains, F1 hybrids and four-way crosses. As a result, in spite of highly heterogeneous population, the LWFD did not show a peculiar response in comparison with four strains and their F1 hybrids. Furthermore, hematological and clinico-biochemical values of the LWFD did not show a large variability as presumed. From these results, it is suggested that hybrids such as four-way crosses among inbred strains can be used as useful animals for the studies of effectiveness and toxicology on drugs.     

Primary structure of guinea-pig Hageman factor: sequence around the cleavage site differs from the human molecule.

The guinea-pig and human Hageman factors differ in their sensitivity to activation by particular bacterial proteinases. To understand this difference, the primary structure and cleavage site on activation of the guinea-pig molecule were determined and compared with the human molecule. By the use of a synthetic oligodeoxyribonucleotide probe which encoded a part of human Hageman factor cDNA, a cDNA clone was isolated from a lambda gt11 cDNA library of guinea-pig liver and sequenced. The cDNA clone was identified as that of guinea-pig Hageman factor by the complete identity of the deduced amino-acid sequence with the actual sequence of the amino-terminal portion of guinea-pig Hageman factor molecule and the active form. The cDNA included part of a leader sequence and the entire coding region of the Hageman factor molecule. Guinea-pig Hageman factor was composed of the same domain structures as the human counterpart with an overall 72% homology in the amino-acid sequence. However, the sequences around the cleavage site were surprisingly different; -Met351-Thr-Arg-Val-Val-Gly-Gly-Leu-Val359-(human) and -Leu338-Ser-Arg-Ile-Val-Gly-Gly-Leu-Val346-(guinea-pig). The amino-acid substitutions around the cleavage site might explain the difference in sensitivity to activation between the human and guinea-pig molecules.     

Design and preclinical testing of an anti-CD41 CAR T cell for the treatment of acute megakaryoblastic leukaemia

Acute megakaryoblastic leukaemia (AMkL) is a rare subtype of acute myeloid leukaemia (AML) representing 5% of all reported cases, and frequently diagnosed in children with Down syndrome. Patients diagnosed with AMkL have low overall survival and have poor outcome to treatment, thus novel therapies such as CAR T cell therapy could represent an alternative in treating AMkL. We investigated the effect of a new CAR T cell which targets CD41, a specific surface antigen for M7-AMkL, against an in vitro model for AMkL, DAMI Luc2 cell line. The performed flow cytometry evaluation highlighted a percentage of 93.8% CAR T cells eGFP-positive and a limited acute effect on lowering the target cell population. However, the interaction between effector and target (E:T) cells, at a low ratio, lowered the cell membrane integrity, and reduced the M7-AMkL cell population after 24 h of co-culture, while the cytotoxic effect was not significant in groups with higher E:T ratio. Our findings suggest that the anti-CD41 CAR T cells are efficient for a limited time spawn and the cytotoxic effect is visible in all experimental groups with low E:T ratio.     

A major difference between the divergence patterns within the lines-1 families in mice and voles

L1 retroposons are represented in mice by subfamilies of interspersed sequences of varied abundance. Previous analyses have indicated that subfamilies are generated by duplicative transposition of a small number of members of the L1 family, the progeny of which then become a major component of the murine L1 population, and are not due to any active processes generating homology within preexisting groups of elements in a particular species. In mice, more than a third of the L1 elements belong to a clade that became active approximately 5 Mya and whose elements are > or = 95% identical. We have collected sequence information from 13 L1 elements isolated from two species of voles (Rodentia: Microtinae: Microtus and Arvicola) and have found that divergence within the vole L1 population is quite different from that in mice, in that there is no abundant subfamily of homologous elements. Individual L1 elements from voles are very divergent from one another and belong to a clade that began a period of elevated duplicative transposition approximately 13 Mya. Sequence analyses of portions of these divergent L1 elements (approximately 250 bp each) gave no evidence for concerted evolution having acted on the vole L1 elements since the split of the two vole lineages approximately 3.5 Mya; that is, the observed interspecific divergence (6.7%-24.7%) is not larger than the intraspecific divergence (7.9%-27.2%), and phylogenetic analyses showed no clustering into Arvicola and Microtus clades.      

Molecular phylogeny and divergence times of drosophilid species

The phylogenetic relationships and divergence times of 39 drosophilid species were studied by using the coding region of the Adh gene. Four genera--Scaptodrosophila, Zaprionus, Drosophila, and Scaptomyza (from Hawaii)--and three Drosophila subgenera--Drosophila, Engiscaptomyza, and Sophophora--were included. After conducting statistical analyses of the nucleotide sequences of the Adh, Adhr (Adh-related gene), and nuclear rRNA genes and a 905-bp segment of mitochondrial DNA, we used Scaptodrosophila as the outgroup. The phylogenetic tree obtained showed that the first major division of drosophilid species occurs between subgenus Sophophora (genus Drosophila) and the group including subgenera Drosophila and Engiscaptomyza plus the genera Zaprionus and Scaptomyza. Subgenus Sophophora is then divided into D. willistoni and the clade of D. obscura and D. melanogaster species groups. In the other major drosophilid group, Zaprionus first separates from the other species, and then D. immigrans leaves the remaining group of species. This remaining group then splits into the D. repleta group and the Hawaiian drosophilid cluster (Hawaiian Drosophila, Engiscaptomyza, and Scaptomyza). Engiscaptomyza and Scaptomyza are tightly clustered. Each of the D. repleta, D. obscura, and D. melanogaster groups is monophyletic. The splitting of subgenera Drosophila and Sophophora apparently occurred about 40 Mya, whereas the D. repleta group and the Hawaiian drosophilid cluster separated about 32 Mya. By contrast, the splitting of Engiscaptomyza and Scaptomyza occurred only about 11 Mya, suggesting that Scaptomyza experienced a rapid morphological evolution. The D. obscura and D. melanogaster groups apparently diverged about 25 Mya. Many of the D. repleta group species studied here have two functional Adh genes (Adh-1 and Adh-2), and these duplicated genes can be explained by two duplication events.      

Chemical structure of the active site of pig heart mitochondrial aspartate aminotransferase labeled with beta-chloro-l-alanine

Formate-induced inactivation of pig heart mitochondrial aspartate aminotransferase by beta-chloro-L-alanine resulted in the modification of the epsilon-amino group of the lysyl residue which is involved in the formation of an aldimine bond with 4-formyl group of the coenzyme, pyridoxal 5'-phosphate. The tryptic peptide isolated from the labeled site of the enzyme was composed of 25 residues and exhibited positive circular dichroism at 325 and 254 nm where the pyridoxyl chromophore of the labeled site peptide absorbs, while the phosphopyridoxyl peptide isolated from the boro-hydride-reduced enzyme did not show any ellipticity in this spectral region. Its comparison with the analogous tryptic peptide from the labeled site of the cytosolic isoenzyme revealed a high degree of homology in their primary structures as well as in spectral properties. Structural analysis of the labeled site peptide and mechanistic consideration of the labeling process indicated that with both isoenzymes the phosphopyridoxyl group is covalently bound to the alpha amino group of the alanyl moiety derived from beta-chloro-L-alanine, the beta carbon of which is covalently linked to the epsilon-amino group of the lysyl residue.