Generation and analysis of mice with a targeted disruption of the arylamine N-acetyltransferase type 2 gene.

Arylamine N-acetyltransferases (NATs) are polymorphic xenobiotic metabolising enzymes, linked to cancer susceptibility in a variety of tissues. In humans and in mice there are multiple NAT isoforms. To identify whether the different isoforms represent inbuilt redundancy or whether they have unique roles, we have generated mice with a null allele of Nat2 by gene targeting. This mouse line conclusively demonstrates that the different isoforms have distinct functions with no compensatory expression in the Nat2 null animals of the other isoforms. In addition, we have used the transgenic line to show the pattern of Nat2 expression during development. Although Nat2 is not essential for embryonic development, it has a widespread tissue distribution from at least embryonic day 9.5. This mouse line now paves the way for the teratological role of Nat2 to be tested.

Arylamine N-acetyltransferases: a pharmacogenomic approach to drug metabolism and endogenous function.

The arylamine N-acetyltransferases (NATs) are a unique family of enzymes that catalyse the transfer of an acetyl group from acetyl-CoA to the terminal nitrogen of hydrazine and arylamine drugs and carcinogens. The NATs have been shown to be important in drug detoxification and carcinogen activation, with humans possessing two isoenzymes encoded by polymorphic genes. This polymorphism has pharmacogenetic implications, leading to different rates of inactivation of drugs, including the anti-tubercular agent isoniazid and the anti-hypertensive drug hydralazine. Mice provide a good model for human NAT, allowing genetic manipulation of expression to explore possible endogenous roles of these enzymes. The first three-dimensional NAT structure was resolved for NAT from Salmonella typhimurium, and subsequently the structure of NAT from Mycobacterium smegmatis has been elucidated. These identified a 'Cys-His-Asp' catalytic triad (conserved in all NATs), which is believed to be responsible for the activation of the active site cysteine residue. As more genomic data become available, NAT homologues continue to be found in prokaryotic species, many of which are pathogenic, including Mycobacterium tuberculosis. The discovery of NAT in M. tuberculosis is particularly significant, since this enzyme participates in inactivation of isoniazid in the bacterium, with implications for isoniazid resistance. Structural studies on NAT proteins and phenotypic analyses of organisms (both mice and prokaryotes) following genetic modifications of the nat genes are leading to an understanding of the potentially diverse roles of NAT in endogenous and xenobiotic metabolism. These studies have indicated that NAT, particularly in Mycobacteria, has the potential to be a drug target. Combinatorial chemical approaches, together with in silico structural studies, will allow for advances in the identification of NAT substrates and inhibitors, both as experimental tools and as potential drugs.

Evidence towards the role of arylamine N-acetyltransferase in Mycobacterium smegmatis and development of a specific antiserum against the homologous enzyme of Mycobacterium tuberculosis.

Arylamine N-acetyltransferase (NAT) in humans inactivates the anti-tubercular drug isoniazid (INH). Homologues of human NAT are present in Mycobacterium tuberculosis and Mycobacterium smegmatis, where they can acetylate, and hence inactivate, INH. The in vivo role of mycobacterial NAT is not known but heterologous expression of the M. tuberculosis gene increases the INH resistance. The 0.85 kb nat gene is part of a gene cluster in M. smegmatis. The gene is transcribed as a large, 7.5 kb mRNA as demonstrated by Northern analysis. A nat knockout strain of M. smegmatis was generated by targeted disruption. The new strain was confirmed to be devoid of NAT activity. The growth of the knockout strain is considerably delayed compared with the wild-type, due to an extended lag phase. The knockout mutant has an increased sensitivity to INH as would be predicted. The NATs from M. smegmatis and M. tuberculosis have a high degree of homology, except in the region of the C terminus. A specific polyclonal antiserum raised against recombinant NAT protein from M. tuberculosis is described that recognizes a stretch of about twenty residues within the C terminus of M. tuberculosis NAT. This highly specific antiserum will enable comparison of nat expression between isolates of M. tuberculosis.

Identification of STKA-dependent genes in Dictyostelium discoideum.

During culmination of Dictyostelium aggregates, prespore and prestalk cells undergo terminal differentiation to form spores and a cellular stalk. Disruption of the cell-fate gene stkA leads to a phenotype in which all the cells destined to become spores end up as stalk cells. 'Stalky' mutants express normal levels of prespore cell transcripts but fail to produce the culmination-stage spore transcript spiA. The stkA gene encodes a putative GATA-type transcription factor (STKA). In order to identify possible downstream targets of STKA we used the technique of mRNA differential display and isolated four cDNA fragments that hybridise to mRNAs present during the later stages of development. All four gene tags were cloned and sequenced. mRNAs represented by these four sequence tags do not accumulate during culmination of 'stalky' cells and therefore must be specific to the spore pathway. By screening a cDNA library, longer cDNAs for all four were cloned and sequenced. Three of these contained complete protein-coding regions while only a partial cDNA was recovered for the fourth. One of the corresponding proteins has significant homology to a surface zinc metalloproteinase (GP63) of the protozoan parasite Leishmania, while another is closely related to a human pre-RNA binding protein (hnRNP R).

Self-efficacy and job-seeking activities in unemployed ethnic youth.

The authors examined variables predicting the self-efficacy and the job-seeking activities of 103 unemployed ethnic youth from diverse cultural backgrounds. Whereas self-esteem and acceptance by the host culture were significant predictors of self-efficacy, the major predictors of job seeking were (a) the extent to which the ethnic youth felt accepted by members of the dominant culture and (b) the difference between their cultural backgrounds and the dominant host culture. Implications of these findings for enhancing the employment of ethnic youth are discussed.

A rapid novel method for the extraction of RNA from wild-type and genetically modified kanamycin resistant mycobacteria.

RNA extraction from mycobacteria can be a difficult and time consuming process due to their unique mycolic acid cell wall composition and the short half life of RNA. Rapid cell wall disruption is essential to isolate intact high quality RNA. Genetically modified Mycobacteria smegmatis resistant to kanamycin demonstrate an altered cell wall composition which impairs the quality of RNA obtained from these mycobacteria. In this report we describe a method of RNA extraction using the detergent Catrimox-14 resulting in high yields of pure, undegraded RNA in less than 1 h. Yields of 25-30 microg RNA per 1x10(9) cells were consistently obtained from both wild-type and genetically modified M. smegmatis. The integrity of this RNA is demonstrated by gel electrophoresis, Northern blot and cDNA analysis.

Phosphorylation of vascular smooth muscle caldesmon by endogenous kinase.

Caldesmon was phosphorylated up to 1.2 molPi/mol using a partially purified endogenous kinase fraction. The phosphorylation site was within the C-terminal 99 amino acids. We were also able to phosphorylate caldesmon incorporated into native and synthetic smooth muscle thin filaments. Phosphorylation did not alter caldesmon binding to actin or inhibition of actomyosin ATPase. It also did not change Ca2+ sensitivity in native thin filaments. Phosphorylated caldesmon bound to myosin less than unphosphorylated caldesmon, especially when the myosin was also not phosphorylated. This work did not support the hypothesis that caldesmon function is modulated by phosphorylation.

Caldesmon binds to smooth muscle myosin and myosin rod and crosslinks thick filaments to actin filaments.

It is well established that caldesmon binds to actin (Kb = 10(7) - 10(-8) M-1) and to tropomyosin (Kb = 10(6) M-1) and that it is a potent inhibitor of actomyosin ATPase. Caldesmon can also bind tightly to myosin. We investigated the binding of smooth muscle and nonmuscle caldesmon isoforms (CDh and CDl respectively) to myosin using proteins from sheep aorta. Both caldesmon isoforms bind to myosin with indistinguishable affinity. The affinity is about 10(6) M-1 in low salt buffer, but is weakened by increasing [KCl] reaching 10(5) M-1 in 100 mM KCl. The stoichiometry of binding is about three caldesmon per myosin molecule. Stoichiometry and affinity are not dependent on whether myosin is phosphorylated nor on the presence of Mg2+ and ATP, provided the ionic strength is maintained constant. The caldesmon binding site of smooth muscle myosin is located in the S-2 region, consequently both HMM and myosin rod bind to caldesmon. Over a range of conditions myosin and myosin rod binding to caldesmon were indistinguishable. Skeletal muscle myosin has no caldesmon binding site. Smooth muscle myosin rods form side-polar filaments in low salt buffer in which the backbone packing of LMM into the filament shaft is clearly visible in negatively-stained electron microscopic images. Sometimes the S-2 portions can be seen 'frayed' from the filament shaft. When caldesmon is bound the filament shaft appears to be about 20% thicker and the frayed effect is dramatically increased; long filamentous 'whiskers' are often seen curving out from the filament shaft. Similar structures are observed with smooth muscle and with non-muscle caldesmon. Myosin also binds to caldesmon when it is incorporated into the thin filament; however, this interaction is qualitatively different. Measurements of smooth muscle HMM binding to native thin filaments in the presence of 3 mM MgATP shows there is a high affinity binding (Kb = 10(6) M-1) which is independent of [Ca2+] and of the level of myosin phosphorylation. The stoichiometry is one HMM molecule per actin monomer which is equivalent to up to 14 HMM bound at high affinity per caldesmon. Negatively stained electron microscopic images of the HMM.ADP.Pi-thin filament complex have failed to show any attachment of HMM to the thin filaments. When rod filaments are added to actin plus caldesmon or to native thin filaments the rod filaments are strongly associated with the actin filament bundles. The majority of rod filaments are lined up parallel and in close proximity to actin filaments.(ABSTRACT TRUNCATED AT 400 WORDS)

Site-directed mutagenesis of arginine 179 of thymidylate synthase. A nonessential substrate-binding residue.

X-ray structural studies have shown that Arg-179 of thymidylate synthase is complexed to bound inorganic phosphate or to the 5'-phosphate of the bound substrate dUMP. The importance of Arg-179 to the structure/function of thymidylate synthase is also indicated by its complete conservation among the 17 thymidylate synthases thus far sequenced. In the present work, Arg-179 has been replaced by Thr, Ala, Lys, and Glu using site-directed mutagenesis with a mixture of four synthetic oligonucleotides as primers. The mutant proteins complement thymidylate synthase-deficient Escherichia coli and show high enzyme activity. Each of these mutants has been purified to homogeneity, partially sequenced to verify the mutation, and has had its steady state kinetic parameters determined. The most significant effect of all mutations is localized to a decrease in the net rate of association of thymidylate synthase with dUMP; the Lys mutant also shows an apparent increase in the dissociation constant of the folate cofactor of the reaction. The high activity in the mutant enzymes is explained by "plasticity" of the enzyme and compensatory actions of the other Arg residues. Why the Arg-179 residue has been conserved during evolution remains an open question.

Electrostatic complementarity within the substrate-binding pocket of trypsin.

The aspartic residue (Asp-189) at the base of the substrate-binding pocket of trypsin was replaced by serine (present in a similar position in chymotrypsin) through site-directed mutagenesis. The wild-type (with Asp-189 in the mature trypsin sequence) and mutant (Ser-189) trypsinogens were expressed in Escherichia coli, purified to homogeneity, activated by enterokinase, and tested with a series of fluorogenic tetrapeptide substrates with the general formula succinyl-Ala-Ala-Pro-Xaa-AMC, where AMC is 7-amino-4-methyl-coumarin and Xaa is Lys, Arg, Tyr, Phe, Leu, or Trp. As compared to [Asp189]trypsin, the activity of [Ser189]trypsin on lysyl and arginyl substrates decreased by about 5 orders of magnitude while its Km values increased only 2- to 6-fold. In contrast, [Ser189]trypsin was 10-50 times more active on the less preferred, chymotrypsin-type substrates (tyrosyl, phenylalanyl, leucyl, and tryptophanyl). The activity of [Ser189]trypsin on lysyl substrate was about 100-fold greater at pH 10.5 than at pH 7.0, indicating that the unprotonated lysine is preferred. Assuming the reaction mechanisms of the wild-type and mutant enzymes to be the same, we calculated the changes in the transition-state energies for various enzyme-substrate pairs to reflect electrostatic and hydrogen-bond interactions. The relative binding energies (E) in the transition state are as follows: EII greater than EPP greater than EPA greater than EIP approximately equal to EIA, where I = ionic, P = nonionic but polar, and A = apolar residues in the binding pocket. These side-chain interactions become prominent during the transition of the Michaelis complex to the tetrahedral transition-state complex.

Cloning, sequencing, and expression of the Lactobacillus casei thymidylate synthase gene.

The thymidylate synthase (TS) gene from Lactobacillus casei has been isolated, cloned, and sequenced. The coding sequence is 948 bp and predicts a primary structure, identical to that reported by protein sequencing methods (Maley et al., 1979b). The gene has been placed in several expression systems which complement TS-deficient Escherichia coli, and express the catalytically active enzyme at levels of 10-20% of the soluble protein of E. coli. The expressed TS has kinetic and structural properties consistent with its being identical to the authentic enzyme from L. casei.

Thermal stability of myosin subfragment-1 decreases upon tryptic digestion in the presence of nucleotides.

Myosin subfragment-1 (S-1), digested with trypsin in the presence of ATP, rapidly loses its ATPase activity upon mild heat treatment even if ATP or ADP is present. The heat-treated molecule is very sensitive to further tryptic digestion. Undigested S-1 and S-1 digested in the absence of ATP are protected by nucleotides. The loss of the protective effect of nucleotides correlates with the tryptic splitting of the 25 kDa amino-terminal fragment between Arg 23 and Ile 24.

Characterization of rabbit masseter muscle fibers.

Myosins of histochemically distinguishable single fibers of rabbit masseter muscle--type 1, 2A, 2B, and slow fibers--have been characterized by gel electrophoresis under dissociating (sodium dodecyl sulfate) and nondissociating (inorganic pyrophosphate) conditions, and by analysis of peptide maps of the heavy chains following limited proteolytic degradation. Type 2B fibers contain more LC3 homodimer than type 2A fibers; peptide maps of their heavy chain are different although the two myosins comigrate on pyrophosphate gel electrophoresis. Slow fiber myosin migrates more slowly than fast myosin and has a distinct peptide map. Differences were also found among fibers of the same histochemical type but originating in different muscles. In adductor magnus 2B myosin the LC1 + LC3 heterodimer band is the strongest, while in masseter 2B myosin the heterodimer is the weakest. Statistical considerations suggest that in masseter there is a mechanism preferentially forming the homodimers. More work is needed to determine the mechanism by which phenotypical differences occur among various fiber types in the same muscle and between corresponding fiber types in different muscles.

Type IIB to IIA fiber transformation in intermittently stimulated rabbit muscles.

Long-term intermittent stimulation (10 Hz, 8 h/day, 7 wk) of the fast-twitch tibialis anterior results in a complete transformation of type IIB fibers to type IIA fibers. This is shown by the histochemical ATPase reaction and by a decrease in Ca2+-uptake ability by the sarcoplasmic reticulum. Furthermore, as shown by studies on bulk myosin and on single fibers, the LC1-to-LC3 light chain ratio is increased on sodium dodecylsulfate gel electrophoretograms, and there are changes in the myosin isozyme pattern manifested on pyrophosphate gels under nondissociating conditions. Thus the staining intensity of the slower moving putative LC1 homodimer band increases, and there is a difference in migration velocity between stimulated and unstimulated isozymes suggesting a possible difference in the heavy chain. This study underlines the importance of the stimulation schedule in determining whether a fast-to-slow transformation or a shift in subtype takes place.

[PCV in newborn babies: a comparison of the hematologic values from the coulter counter with the centrifuged PCV (author's transl)]. / Hämatokritbestimmung beim Neugeborenen: Vergleich zwischen der Coulterschen Methode und dem zentrifugierten Wert.

The packed cell volume (PCV) is an essential index in the evaluation of distressed newborn babies and often allows an early diagnosis of a hyperviscosity syndrome to be made. The PCV can be measured easily and very accurately by centrifuging a heelprick blood sample in a heparinized glass capillary (PCV-c). Commonly the red blood picture including PCV is determined automatically by means of a Coulter Counter (CC) thus allowing the increasing demands of a bigger unit to be coped with. The comparison of the hematologic values (red coll count, Hgb, PCV) as given by the CC, with the PCV-c shows an excellent correlation between red cell count, Hgb and PCV-c and no statistically significant difference of the mean PCV-values of both methods. However, as the PCV-c proves to be more reliable, a wide scattering of the individual PCV-values obtained by the CC is found. Furthermore there is a marked tendency to lower PCV-readings by the CC in the critical zone above 68%, eventually leading to a delay in the diagnosis of a hyperviscosity syndrome and therefore, at least in neonatology, the PCV-c should be preferred.

A kinetic study of the K+ activated ATPase of myosin subfragment-1.

The K+ activated ATPase activity of myosin subfragment-1 was measured under different conditions and enzyme kinetic parameters were calculated. The logarithm of Km varies linearly with ionic strength down to very low KCl concentrations, the logarithm of k2 vs. ionic strength, on the other hand, considerably deviates from linearity at low concentrations of KCl. ADP is a competitive inhibitor, like myosin ATPase, and with practically the same inhibitor constant. The energetical parameters of the decomposition (to products and enzyme) of the S-1--ATP complex are partically the same as those for myosin, the parameters of its formation, however, differ from the corresponding values for myosin: delta SI is considerably, delta HI significantly higher in the case of myosin. This may be the result of some kind of interaction of the two heads of myosin.

A simple procedure for the preparation of electrophoretically homogeneous alpha-actin from rabbit muscle.

A simplified procedure for the preparation of alpha-actinin from rabbit skeletal muscle is described. Use is made of the observation that actin is completely eliminated by denaturation, when myofibrils are dissolved in 0.6 M MgCl2 and thereafter MgCl2 is removed by dialysis.