ε-caprolactam,a new powerful inducer for the formation of Rhodococcus rhodochrous J1 nitrilase

We found that -caprolactam is a new powerful inducer for the formation of Rhodococcus rhodochrous J1 nitrilase. When Rhodococcus rhodochrous J1 cells were cultivated at 28°C for 120 h in a nutrient medium supplemented with 0.5% (w/v) -caprolactam, an enormous amount of nitrilase was formed in the cells which corresponded to approximately 30% of all soluble protein. The level of -caprolactam in the culture broth barely decreased in the course of cultivation. -Butyrolactam and -valerolactam also caused effective induction. The induction of nitrilase formation by -caprolactam was also observed in some other Rhodococcus strains.     

Pyridoxal-5′-phosphate derivatives of substance P: Preparation,spasmogenic activity,and degradation by human plasma

Two fluorescent derivatives of substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂) were prepared by chemical modification of the native peptide by pyridoxal-5-phosphate (pyridoxal-P). The formation of both pyridoxal-P-derivatives of SP is the result of one modification procedure. The determination of the amino acid composition showed that in one of the derivatives the -amino group of the Lys residue [-(P-pxy)-SP] and in the other the -amino group of the Lys residue and also the N-terminal amino group [,-di-(P-pxy)-SP] of SP had been substituted by pyridoxal-P. -(P-pxy)-SP and ,-di-(P-pxy)-SP have spasmogenic activity with ED₅₀ of 1.8×10^–9 and 4×10^–9 M, respectively, tested on isolated guinea pig ileum. The fluorescence of P-pxy residues permits detection of as little as 1 pmol/ml of -(P-pxy)-SP and 0.5 pmol/ml of ,-di-(P-pxy)-SP. Both analogues of SP obtained are degraded by human plasma more slowly than the native peptide.Abbreviations SP substance P - pyridoxal-P pyridoxal-5-phosphate - P-pxy phospho-pyridoxyl residue - -(P-pxy)-SP substance P modified by pyridoxal-P at the -amino group of the Lys residue - ,-di-(P-pxy)-SP substance P modified by pyridoxal-P at the -amino group of the Lys residue and the N-terminal amino group of SP - (P-pxy)-Lys Lys modified by pyridoxal-P at the -amino group     

Substitutions of the Conserved Thr42 Increased the Roles of the ε-Subunit of Maize CF1 as CF1 Inhibitor and Proton Gate

The conserved residue Thr42 of -subunit of the chloroplast ATP synthase of maize (Zea mays L.) was substituted with Cys, Arg, and Ile, respectively, through site-directed mutagenesis. The over-expressed and refolded -proteins were purified by chromatography on DEAE-cellulose and FPLC on mono-Q column, which were as biologically active (inhibiting Ca²⁺-ATPase activity and blocking proton gate) as the native subunit isolated from chloroplasts. The T42C and T42R showed higher inhibitory activities on the soluble CF₁(–) Ca²⁺-ATPase than the WT. The T42I inhibited the Ca²⁺-ATPase activity of soluble CF₁ and restored photophosphorylation activity of membrane-bound CF₁ deficient in the most efficiently. Far-ultraviolet CD spectra showed that the portions of -helix and -sheet structures of the three mutants were somewhat different from WT. Thus the conserved residue Thr42 may be important for maintaining the structure and function of the -subunit and the basic functions of the -subunit as far as an inhibitor of Ca²⁺-ATPase and the proton gate are related.     

The chloroplast ATP synthase: Structural changes during catalysis

This article summarizes some of the evidence for the existence of light-driven structural changes in the and subunits of the chlorplast ATP synthase. Formation of a transmembrane proton gradient results in: (1) a change in the position of the subunit such that it becomes exposed to polyclonal antibodies and to reagents which selectively modifyLys109; (2) enhanced solvent accessibility of several sulfhydryl residues on the subunit; and (3) release/ exchange of tightly bound ADP from the enzyme. These and related experimental observations can, at least partially, be explained in terms of two different bound conformational states of the subunit. Evidence for structural changes in the enzyme which are driven by light or nucleotide binding is discussed with special reference to the popular rotational model for catalysis.     

DNA polymerase III holoenzyme of Escherichia coli: Components and function of a true replicative complex

Summary The DNA polymerase III holoenzyme is a complex, multisubunit enzyme that is responsible for the synthesis of most of the Escherichia coli chromosome. Through studies of the structure, function and regulation of this enzyme over the past decade, considerable progress has been made in the understanding of the features of a true replicative complex. The holoenzyme contains at least seven different subunits. Three of these, , and , compose the catalytic core. Apparently is the catalytic subunit and the product of the dnaE gene. Epsilon, encoded by dnaQ (mutD), is responsible for the proofreading 35 activity of the polymerase. The function of the B subunit remains to be established. The auxiliary subunits, , and , encoded by dnaN, dnaZ and dnaX, respectively, are required for the functioning of the polymerase on natural chromosomes. All of the proteins participate in increasing the processivity of the polymerase and in the ATP-dependent formation of an initiation complex. Tau causes the polymerase to dimerize, perhaps forming a structure that can coordinate leading and lagging strand synthesis at the replication fork. This dimeric complex may be asymmetric with properties consistent with the distinct requirements for leading and lagging strand synthesis.     

Kinetic comparison of caiman ε-crystallin and authentic lactate dehydrogenases of vertebrates

Kinetic comparison of -crystallins isolated from the avian and reptilian species and the authentic lactate dehydrogenases (LDHs) was undertaken in order to clarify the identities of these structural lens proteins in relation to their enzymatic activity. Caiman -crystallin similar to the previously characterized duck -crystallin appeared to possess a genuine and stable LDH activity as detected by nitro blue tetrazolium staining on polyacrylamide gels and conventional kinetic assays. Kinetic parameters for pyruvate,l-lactate, NAD⁺, and three structural analogues of the coenzyme in this -crystallin catalyzed reaction were also determined and compared. Despite the structural similarities between -crystallins and chicken heart LDH, differences in charge and kinetic properties have been revealed by native isozyme electrophoresis and kinetic analysis as examined by initial velocity and substrate inhibition studies. It is found that the kinetic data analyzed for caiman -crystallin were more fitted with a compulsory ordered Bi-Bi sequential mechanism similar to those for the authentic LDHs and duck -crystallin. Caiman -crystallin has for the first time been established as a heart-type LDH based on the kinetic analysis and comparison with the authentic heart- and muscle-type LDHs from pig and chicken.     

Intracellular sites of the synthesis of sweet potato mitochondrial F1ATPase subunits

Summary Five subunits (-, -, -, - and -subunits) of the six -and -subunits) in the F₁ portion (F₁ATPase) of sweet potato (Ipomoea batatas) mitochondrial adenosine triphosphatase were isolated by an electrophoretic method. The - and -subunits were not distinguishable immunologically but showed completely different tryptic peptide maps, indicating that they were different molecular species. In vitro protein synthesis with isolated sweet potato root mitochondria produced only the -subunit when analyzed with anti-sweet potato F₁ATPase antibody reacting with all the subunits except the -subunit. Sweet potato root poly(A)⁺RNA directed the synthesis of six polypeptides which were immunoprecipitated by the antibody: two of them immunologically related to the -subunit and the others to the - and -subunits. We conclude that the -subunit of the F₁ATPase is synthesized only in the mitochondria and the -, - and -subunits are in the cytoplasm.     

Interaction of Apolipoprotein E ε 4 With Other Genetic and Non-Genetic Risk Factors in Late Onset Alzheimer Disease: Problems Facing the Investigator

The Apolipoprotein E4 allele (Apo-4) is the major susceptibility gene for late onset Alzheimer Disease (AD) but epidemiological data suggest that the effect of this allele is modified in different individuals by genetic or environmental factors. Age and head injury are major non-genetic factors modifying the Apo-4 risk. There is conflicting data as to whether alleles of other chaperon proteins (such as alpha-1-antichymotrypsin (ACT)) or Apo-4 receptors (such as the VDRL receptor) modify the Apo-4 risk for AD. We analyze problems posed by genetic association studies including those of multiple comparisons and selection of controls, the latter problem exacerbated by the wide variations in Apolipoprotein E allele frequencies observed in different groups and localities.     

Detection of ε(γ-glutamyl) lysine

Summary Detection of (-glutamyl) lysine crosslinks is not only necessary for establishing the importance of the dipeptide as a post-translational modification of proteins, but provides information as to the importance of the transglutaminase enzyme in a biological system. The crosslink may be detected using both indirect and direct methodology. Indirect methods for its detection include measurement of masked lysines within a protein, detection of polymer formation by gel-electrophoresis and the inhibition of crosslinking by the incorporation of small molecular weight amines into the substrate protein. Direct methods for the detection of (-glutamyl) lysine require the actual isolation of the dipeptide following its release from the sample protein by exhaustive proteolytic digestion. Separation of the dipeptide from other components of the digest may be achieved by either ion-exchange chromatography or gel filtration and its qualitative identification achieved by techniques such as paper-electrophoresis or thin layer chromatography. Quantitative estimation of (-glutamyl) lysine normally involves its further separation by ion-exchange chromatography and its post-column detection following derivatisation with ninhydrin. More recent techniques include pre-column derivatisation of the dipeptide with fluorogenic reagents such as -pthalaldehyde and separation by reverse phase HPLC. With the recent advances in liquid chromatography resulting in the improved resolution of amino acids, increased sensitivity, rapid analysis times, and small sample sizes, it appears likely that direct quantitation of (-glutamyl) lysine will be the preferred method for the future.     

Lysine Dendrimers and Their Starburst Polymer Derivatives: Possible Application for DNA Compaction and in vitro Delivery of Genetic Constructs

We attempted to find some compounds for the effective delivery of gene constructs into cells and obtained two trispherical dendrimers on the basis of lysine, (Lys)₈-(,-Lys)₄-(,-Lys)₂-(,-Lys)-Ala-NH₂ (D1) and ((Lys)₈-(,-Lys)₄-(,-Lys)₂-,-Lys)-Ala-[Lys(Plm)]₂-Ala-NH₂ (D2), as well as the starburst polymeric derivatives of D1, (pVIm) ₈ -D1 and (pLys) _n -D1, containing poly(N-vinylimidazole) and polylysine chains single-point bound to the dendrimer amino groups. The conditions of dendrimer–plasmid DNA complex formation were studied. The intracellular localization of these complexes and the expression of gene constructs delivered with their help were analyzed in transfection experiments on the HeLa cell cultures of human epithelial carcinoma and on mouse C2C12 myoblasts. It was found that the chemical structure of dendrimer D1 and its derivatives significantly affected the structure and properties of complex.     

Polymorphism of the Apolipoprotein E Gene and Risk of Myocardial Infarction

The apolipoprotein E (ApoE) gene polymorphism resulting from nucleotide substitutions in exon 4 was analyzed in Russian and Tatar patients with myocardial infarction (MI) from Bashkortostan. Alleles 2, 3, and 4 were identified by PCR. The genotype frequency distribution proved to be age-dependent in healthy Russians, genotype 2/3 increasing in frequency in subjects over 45. Russians who suffered MI under 45 had lower frequencies of genotype E3/3 (50.00% vs. 75.47% in controls of the same age, = 0.013, OR = 0.33) and allele 3 (72.12% vs. 85.85%, = 0.020, OR = 0.43) and a higher frequency of allele 4 (22.12% vs. 10.38%, = 0.030, OR = 2.45). Russians who suffered MI complicated by cardiogenic shock (CS) had a significantly higher frequency of genotype 3/4 and lower frequencies of genotype 3/3 and allele 3 as compared with MI patients without CS. In Tatars, genotype 4/4 occurred at a frequency of 14.29% in patients who suffered MI under 45, and was not detected in healthy subjects of the same age ( = 0.024, OR = 17.85). Thus, the ApoE polymorphism was associated with higher risk of MI in Russians and Tatars under 45.     

Polynucleotides and Their Components in the Processes of Aromatic Nucleophilic Substitution: II.1 Nucleophilic Modification of 3′,5′-Bis-O- (α,β,α′,β′-tetrafluoropyrid-γ-yl)thymidine

The interaction of 3,5-bis-O-(,,,-tetrafluoropyrid--yl)thymidine with various nucleophilic reagents was studied to evaluate the possibility of molecular design of new types of nucleic acid analogues using S _NAr reactions. The reactions with morpholine and sodium azide led to the introduction of one and two nucleophilic residues into each of the polyfluorinated pyridine rings. The nucleophilic polycondensation with bifunctional reagents ethylenediamine and hexamethylenediamine depended on the nature of nucleophile and reaction conditions and resulted in the formation of supramolecules containing about five or more than 20 pyrimidine bases.     

Assignment of aliphatic side-chain 1HN/15N resonances in perdeuterated proteins

Summary The perdeuteration of aliphatic sites in large proteins has been shown to greatly facilitate the process of sequential backbone and side-chain ¹³C assignments and has also been utilized in obtaining long-range NOE distance restraints for structure calculations. To obtain the maximum information from a 4D ¹⁵N/¹⁵N-separated NOESY, as many main-chain and side-chain ¹H_N/¹⁵N resonances as possible must be assigned. Traditionally, only backbone amide ¹H_N/¹⁵N resonances are assigned by correlation experiments, whereas slowly exchanging side-chain amide, amino, and guanidino protons are assigned by NOEs to side-chain aliphatic protons. In a perdeuterated protein, however, there is a minimal number of such protons. We have therefore developed several gradient-enhanced and sensitivity-enhanced pulse sequences, containing water-flipback pulses, to provide through-bond correlations of the aliphatic side-chain ¹H_N/¹⁵N resonances to side-chain ¹³C resonances with high sensitivity: NH₂-filtered 2D ¹H-¹⁵N HSQC (H₂N-HSQC), 3D H₂N(CO)C_/ and 3D H₂N(COC_/)C_/ for glutamine and asparagine side-chain amide groups; 2D refocused H(N_/)C_/ and H(N_/C_/)C_/ for arginine side-chain amino groups and non-refocused versions for lysine side-chain amino groups; and 2D refocused H(N)C and nonrefocused H(N_.)C for arginine side-chain guanidino groups. These pulse sequences have been applied to perdeuterated ¹³C-/¹⁵N-labeled human carbonic anhydrase II (²H-HCA II). Because more than 95% of all side-chain ¹³C resonances in ²H-HCA II have already been assigned with the C(CC)(CO)NH experiment, the assignment of the side-chain ¹H_N/¹⁵N resonances has been straightforward using the pulse sequences mentioned above. The importance of assigning these side-chain H_N protons has been demonstrated by recent studies in which the calculation of protein global folds was simulated using only ¹H_N-¹H_N NOE restraints. In these studies, the inclusion of NOE restraints to side-chain H_N protons significantly improved the quality of the global fold that could be determined for a perdeuterated protein [R.A. Venters et al. (1995) J. Am. Chem. Soc., 117, 9592–9593].To whom correspondence should be addressed.     

Molecular organization of the human CD3 gene family on chromosome 11q23

The genes encoding three invariant components of the human T-cell antigen receptor, the CD3 , , and chains, are located on human chromosome 11 at band q23. We isolated cosmid clones containing the human CD3 and chain genes in vectors designed for rapid and efficient chromosome walking. The human CD3 gene was located in the region immediately downstream of the CD3 and genes using synthetic oligonucleotide probes and the localization of this gene confirmed by DNA sequencing. Detailed restriction mapping of the CD3 locus demonstrated that all three CD3 subunits are encoded within 60 kb of DNA with the CD3 gene located 26 kb downstream of the CD3 and genes. Analysis of genomic DNA on pulsed field gels using probes isolated from these cosmid clones defined a physical map of 750 kb spanning the CD3 locus on human chromosome 11g23. The CD3 genes thus comprise a multigene family encoding cell surface components important for transmembrane signaling on T lymphocytes. The arrangement of these genes suggest that they may share common regulatory elements for the control of gene expression during T-cell ontogeny.     

Modeling the growth curve for Spirulina (Arthrospira) maxima, a versatile microalga for producing uniformly labelled compounds with stable isotopes

The paper presents a five-phase model to describe batch culture of Spirulina maxima under limitations of light and nutrients nitrogen andsulfur. The general equation for the exponential, linear, decelerating andstationary phases of the growth curve took into account that (i) the specificgrowth rate was proportional to the local light intensity in thephotobioreactor; (ii) light attenuation was due to cell's absorption andreflection and observed the Lambert-Beer's law with a total absorptioncoefficient (_T) that was the sum of absorption coefficient(_a) and reflection coefficient (_r); (iii) thecomposition of the alga and its absorption _a changed withtime and (iv) the specific growth rate was influenced by nutrients ofnitrogen and sulfur in the culture medium according to a Monod's law.Two successive equations describing the death phase were proposed basedon the hypothesis that the death rate accelerated with the %PSC until itreached its maximum. From that point the %PSC decreased and the deathrate reduced. Our model fitted very well the cultures grown in the photobioreactor developed in our laboratory.     

Synthesis of Nα-(fluoren-9-ylmethoxycarbonyl)-Nε-[(7-methoxycoumarin-4-yl)acetyl]-L-lysine for use in solid-phase synthesis of fluorogenic substrates

N -(fluoren-9-ylmethoxycarbonyl)-N -[(7-methoxycoumarin-4-yl)acetyl]-L-lysine[Fmoc-Lys(Mca)-OH] has been conveniently prepared. The copper complex of L-lysine hydrochloride was initially prepared, followed by the addition of the (7-methoxycoumarin-4-yl)acetyl (Mca) group to the -amino group. The copper complex was decomposed, and the Fmoc group was introduced to the -amino group. Fmoc-Lys(Mca)-OH was recrystallized from hexane and could be used directly for the solid-phase synthesis of fluorogenic substrates.     

Locus assignment of human α-globin structural mutants by selective enzymatic amplification of α1 and α2-globin cDNAs

Summary We have used the powerful methodology of DNA enzymatic amplification in order to assign human -globin structural mutants to one of the two highly homologous -globin genes. Selectively amplified 1 and 2-globin cDNAs were dot-blotted and further hybridized to synthetic oligonucleotides encompassing either the normal or the mutated sequences. The generated signals corresponded specifically to one of the two -globin genes. Using this approach the -globin structural mutants J-Buda and G-Pest were found to be encoded by the 2 and the 1-globin genes, respectively. Furthermore, the exact nucleotide changes were determined. We propose this technique to serve as a simple and definitive method for assigning -globin structural mutants.     

The structural features of effective antagonists of the luteinizing hormone releasing hormone

Summary The structure-activity data of 6 years on 395 analogs of the luteinizing hormone releasing hormone (LHRH) have been studied to determine effective substituents for the ten positions for maximal antiovulatory activity and minimal histamine release. The numbers of substituents studied in the ten positions are as follows: (41)¹-(12)²-(12)³-(5)⁴-(47)⁵-(52)⁶-(16)⁷-(18)⁸-(4)⁹-(8)¹⁰. In position 1, DNal and DQal were effective with the former being more frequently the better substituent. DpClPhe was uniquely effective in position 2. Positions 3 and 4 are very sensitive to change. D3Pal in position 3 and Ser in position 4 of LHRH were in the best antagonists. PicLys and cPzACAla were the most successful residues in position 5 with cPzACAla being the better substituent. Position 6 was the most flexible and many substituents were effective; particularly DPicLys. Leu⁷ was most often present in the best antagonists. In position 8, Arg was effective for both antiovulatory activity and histamine release; ILys was effective for potency and lesser histamine release. Pro⁹ of LHRH was retained. DAlaNH₂ ¹⁰ was in the best antagonists.Abbreviations AABLys N -(4-acetylaminobenzoyl)lysine - AALys N -anisinoyl-lysine - AAPhe 3-(4-acetylaminophenyl)lysine - Abu 2-aminobutyric acid - ACLys N -(6-aminocaproyl)lysine - ACyh 1-aminocyclohexanecarboxylic acid - ACyp 1-aminocyclopentanecarboxylic acid - Aile alloisoleucine - AnGlu 4-(4-methoxy-phenylcarbamoyl)-2-aminobutyric acid - 2ANic 2-aminonicotinic acid - 6ANic 6-aminonicotinic acid - APic 6-aminopicolinic acid - APh 4-aminobenzoic acid - APhe 4-aminophynylalanine - APz 3-amino-2-pyrazinecarboxylic acid - Aze azetidine-2-carboxylic acid - Bim 5-benzimidazolecarboxylic acid - BzLys N -benzoyllysine - Cit citrulline - Cl₂Phe 3-(3,4-dichlorphenyl)alanine - cPzACAla cis-3-(4-pyrazinylcarbonylaminocyclohexyl)alnine - cPmACAla cis-3-[4-(4-pyrimidylcarbonyl)aminocyclohexyl]alanine - Dbf 3-(2-dibenzofuranyl)alanine - DMGLys N -(N,N-dimethylglycyl)lysine - Dpo N -(4,6-dimethyl-2-pyrimidyl)-ornithine - F₂Ala 3,3-difluoroalanine - hNal 4-(2-naphthyl)-2-aminobutyric acid - HOBLys N -(4-hydroxybenzoyl)lysine - hpClPhe 4-(4-chlorophenyl)-2-amino-butyric acid - Hse homoserine, 2-amino-4-hydroxybutanoic acid - ICapLys N -(6-isopropylaminocaproyl)lysine - ILys N -isopropyllysine - Ind indoline-2-carboxylic acid - INicLys N -isonicotinoyllysine - IOrn N -isopropylornithine - Me₃Arg N^G,N^G,N^G-trimethylarginine - Me₂Lys N ,N -dimethyllysine - MNal 3-[(6-methyl)-2-naphtyl]alanine - MNicLys N -(6-methylpicolinoyl)lysine - MPicLys N -(6-methylpicolinoyl)lysine - MOB 4-methoxybenzoyl - MpClPhe N-methyl-3-(4-chlorphenyl)lysine - MPZGlu glutamic acid,-4-methylpiperazine - Nal 3-(2-naphthyl)alanine - Nap 2-naphthoic acid - NicLys N -nicotinoyllysine - NO₂B 4-nitrobenzoyl - NO₂Phe 3-(4-nitrophenyl)alanine - oClPhe 3-(2-chlorphenyl)alanine - Opt O-phenyl-tyrosine - Pal 3-(3-pyridyl)alanine - 2Pal 3-(2-pyridyl)alanine - 2PALys N -(3-pyridylacetyl)lysine - pCapLys N -(6-picolinoylaminocaproyl)lysine - pClPhe 3-(4-chlorophenyl)alanine - pFPhe 3-(4-fluorophenyl)-alanine - Pic picolinic acid - PicLys N -picolinoyllysine - Pip piperidine-2-car-boxylic acid - PmcLys N -(4-pyrimidylcarbonyl)lysine - Ptf 3-(4-trifluromethyl phenyl)alanine - Pz pyrazinecarboxylic acid - PzAla 3-pyrazinylalanine - PzAPhe 3-(4-pyrazinylcarbonylaminophenyl)alanine - Qal 3-(3-quinolyl)alanine - Qnd-Lys N -quinaldoyllysine - Qui 3-quinolinecarboxylic acid - Qux 2-quinoxalinecarboxylic acid - Tic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid - TinGly 2-thienylglycine - tNACAla trans-3-(4-nicotinoylaminocyclohexyl)-alanine - tPACAla trans-3-(4-picolinoylaminocyclohexyl)alanine     

Transmission of primary trisomics in pearl millet

Summary Transmission rates of extra chromosomes found in the full set of trisomics of pearl millet (Pennisetum americanum) (2n = 14) were estimated by examining the progeny of selfed trisomics and the progeny of trisomics crossed to disomics. When the trisomics were selfed, dark green and tiny had the highest transmission rate (23.8% and 23.3%, respectively) and pseudonormal the lowest (13.8%). Other trisomics had an intermediate rate of transmission. When the trisomics were used as females in crosses with disomics, both dark green and tiny again had the highest transmission rate and pseudonormal the lowest. When the trisomics were used as males in crosses to disomics, no trisomic was transmitted to the progeny except for spindle, and this occurred with a very low frequency (2.0%). A variation in transmission rate was observed from plant to plant and season to season for the same trisomic type. A study of the transmission rate of the extra chromosomes indicated that the following factors were probably contributing to the lower rate of transmission: small- or light-weight seeds tended to have a higher proportion of trisomics than heavier seeds; lighter seeds had a lower percentage germination; a positive and significant correlation was noticed between trivalent frequency and transmission rate. Plants with reduced vigour produced a higher frequency of trisomics. Though trisomics involving longer extra chromosomes showed a high degree of pollen and ovule sterility, they were highly transmissible. This has resulted in a close relationship between gametic sterility and transmission rate of extra chromosome.