A solid-phase synthesis of three aza-, iminoaza- and reduced aza-peptides from the same precursor

Using the Boc-strategy, a step-by-step synthesis on the PAM solid supportof three aza-, iminoaza- and reduced aza-peptide homologues is described.From the same hydrazinocarbonyl peptide-PAM precursor, the coupling ofeither a Boc-amino acid or a Boc-amino aldehyde gives rise to an aza-peptideor an iminoaza-peptide containing theC-CO-NH-N-CO-NH-C orC-CH=N-N-CO-NH-C surrogate of the peptide motif, respectively. In situreduction of the latter by NaBH₃CN leads to a reducedaza-peptide containing theC-CH₂-NH-N-CO-NH-C moiety. The key step synthesis of thehydrazinocarbonyl peptide-PAM precursor is carried out by coupling on thegrowing peptide chain the N-Boc-aza-amino acid chloride obtained by theaction of triphosgene on the corresponding N-Boc-hydrazine. Thesemodifications have been introduced in position 1-2 of the YLGYLEQLLRbenzodiazepine-like decapeptide     

Resistance of α-globulin fromSesamum indicum L. to proteases in relationship to its structure

-Globulin, the high-molecular-weight protein fraction fromSesamum indicum L., was hydrolyzed to low-molecular-weight protein and peptides by pepsin, while its resistance to hydrolysis by group-specific enzymes, trypsin or -chymotrypsin, was very high. The protein showed definite structural changes after proteolysis, especially after peptic hydrolysis, as evidenced from various biophysical data. The sedimentation velocity pattern of -globulin hydrolyzed by trypsin or -chymotrypsin indicated reduction in the percentage of 11S component, while the pepsinhydrolyzed sample was devoid of any 11S component, indicating the absence of a native protein molecule. The fluorescence emission spectra of the various hydrolyzed -globulin showed a red shift in the fluorescence emission maximum. The red shift was maximum with -globulin hydrolyzed by pepsin and minimum with the trypsin-hydrolyzed sample. The far-ultraviolet-circular dichroic measurements indicated that most of the ordered structure of -globulin was absent after pepsin hydrolysis, while after trypsin and chymotrypsin hydrolysis conformational changes were less.     

α-Thalassemia and the production of different α chain variants in heterozygotes

The production of five chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average / values of about 1.1. They were assigned a normal genotype (^G/), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low ^G/ values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher ^G/ values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate chain deficiency due to an -thalassemia-2 heterozygosity (^G/⁰ or ^0G/) or a homozygosity (^0G/⁰), respectively.This study was supported in part by USPHS Research Grants HLB-05168 and HLB-15158.     

Alpha-thalassemia in Papua New Guinea

Summary A study of the distribution of -thalassemia in Papua New Guinea (PNG) was carried out by DNA analysis. A total of 664 DNA samples were screened for -thalassemia 2 and -thalassemia 1 caused respectively by either deletion of one or both of the duplicated -globin genes. -Thalassemia 2 was detected in high frequencies in coastal and lowland regions where malaria has been holo- to hyperendemic but in low frequencies in non-malarious highland regions. The highest frequency was observed in the north coast of PNG. The distribution of -thalassemia 2 seems to be in accordance with other conditions such as ovalocytosis and G6PD deficiency which are also prevalent in this population, suggesting that they may interact in protection against malaria. However, it appears to be negatively correlated with -thalassemia and -thalassemia 1, the latter being extremely rare in this population. Analysis of the types and subtypes of the single -globin gene deletion revealed a predominance of the –^4.2 type in general, except in some regions in the south where the –^3.7 type is prevalent. The –^3.7 I subtype is the common form of the –^3.7 deletion in the PNG mainland. The –^3.7 III subtype, previously reported to be unique in Melanesians and Polynesians, was detected in an offshore island of PNG. However, this subtype is very rare in Melanesians from the PNG mainland.     

Acetate inhibition on growth of recombinant E. coli and expression of fusion protein TGFaα-PE40

Summary Acetate was inhibitory to the growth of early induced E. coli cells and their expression of fusion protein, transforming growth factor--Pseudomonas exotoxin 40 (TGF-PE40), but the inhibitory level was strain dependent For E. coli JM109 (pTAC-TGF57-PE40), 2 g/L of added acetate (3 g/L of total acetate in the medium) decreased TGFa-PE40 production by 38.0%. Acetate was less inhibitory to E. coli RR1, and RR1 was not affected by adding 2 g/L of acetate. However, 5 g/L of added acetate (6.7 g/L of total acetate in the medium) decreased TGF-PE40 production by 21.2%. These results indicate that higher acetate concentration was associated with inhibition of TGF-PE40 expression of E. coli JM109 during late induction.     

Tumor necrosis factor α modifies resistance to interferon α in vivo: First clinical data

Summary Patients with Philadelphia-positive chronic-phase chronic myelogenous leukemia (CML) resistant to interferon (IFN) were treated in a phase I/II study with recombinant human tumor necrosis factor to overcome IFN resistance. Doses of 40, 80, 120 or 160 µg/m² TNF were given as 2-h infusions on 5 consecutive days every 3 weeks. IFN (4 × 10⁶ IU/m² s.c., daily) treatment was continued. Six patients were treated, completing 1–24 (median, 12) treatment cycles. Five of the six patients achieved partial hematological remission, while the remaining patient had to stop treatment because of WHO grade 4 thrombocytopenia following the first TNF cycle. No complete hematologic remission or cytogenetic improvement was seen. Side-effects were similar to those described for both substances alone. Maximum tolerable TNF doses usually varied between 80 µg/m² and 160 µg/m². To examine possible pathways of TNF activity in these patients, interferon receptor status and (2–5)-oligoadenylate synthetase levels were examined in peripheral blood mononuclear cells. Both parameters remained unchanged during TNF treatment. These preliminary data point to significant clinical efficacy of additionally applyed TNF in IFN-resistant CML patients.     

TROSY experiment for refinement of backbone psi and phi by simultaneous measurements of cross-correlated relaxation rates and 3,4J(H alpha HN) coupling constants

The TROSY principle has been introduced into a HNCA experiment, which is designed for measurements of the intraresidual and sequential H-C/H^N-N dipole/dipole and H-C/N dipole/CSA cross-correlated relaxation rates. In addition, the new experiment provides values of the ^3,4 J _{H HN} coupling constants measured in an E.COSY manner. The conformational restraints for the and angles are obtained through the use of the cross-correlated relaxation rates together with the Karplus-type dependencies of the coupling constants. Improved signal-to-noise is achieved through preservation of all coherence transfer pathways and application of the TROSY principle. The application of the [¹⁵N,¹³C]-DQ/ZQ-[¹⁵N,¹H]-TROSY-E.COSY experiment to the 16 kDa apo-form of the E. coli Heme Chaperon protein CcmE is described. Overall good agreement is achieved between and angles measured with the new experiment and the average values determined from an ensemble of 20 NMR conformers.     

Cell wall anionic polymers and peptidoglycan of Actinoplanes philippinensis VKM Ac-647

The cell wall of Actinoplanes philippinesis VKM Ac-647 harbours several carbohydrate-containing anionic polymers. (1) The main polymer of the wall is of a poly(glycosylglycerol phosphate) nature. Its monomeric units — O--d-mannopyranosyl-(14)--d-galactopyranosyl-(11)-glycerol monophosphates — are connected by phosphodiester bonds involving the hydroxyl groups at glycerol C3 and galactose C6. There also are chains without mannosyl substitutents. The teichoic acid structure has been established by chemical analysis and with ¹H and ¹³C NMR spectroscopy. This is the first finding of a teichoic acid with mannosyl residues in a bacterial cell wall. (2) The phosphorylated mannan contains mannose and 2-O-methylmannose. Its core chain has -1,2; -1,3; and -1,6 substitutions as revealed by ¹³C NMR spectroscopy.The peptide unit of the peptidoglycan contains no l-alanine, instead of which position 1 is occupied by glycine; and diaminopimelic acid is represented, besides its meso- (or DD) form, by small amounts of its LL isomer.Abbreviations Gro glycerol - Gro2P glycerol-2 phosphate - APT attached-proton-test - P_tot total content of phosphorus - P_lab phosphorus mineralized in 7 min at 100°C - P_NA phosphorus of nucleic acids - P_stab stable phosphorus - T trace amounts     

Hexahydroindanone derivatives of steroids formed by Rhodococcus equi

Summary A mutant strain of Rhodococcus equi accumulates three metabolites from the androst-4-ene-3,17-dione or from its degradation intermediate, 3a-H-4(3'-propionic acid)-7a-methylhexahydro-1,5-indanedione (MEPHIP). These three metabolites are: 3a-H-4a(3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone--lactone (HIL); 3a-H-4(3'-trans acrylic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (^2'-5-hydroxy-MEPHIP); and 3a-H-4(3'-hydroxy-3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (3'-hydroxy-HIL). The behaviour of this mutant allows us to propose a pathway for degradation of the intermediates, methylperhydroindanone propionates. However, during this degradation, the side-chain propionate was eliminated by a-oxidation mechanism. Offprint requests to: A. Miclo     

The α1 and α2 domains of H-2 class I molecules interact to form unique epitopes

Mouse class I antigens are the major targets of cytolytic T lymphocytes in both major histocompatibility complex (MHC)-restricted and allogeneic responses. Considerable evidence has recently accumulated demonstrating that MHC class I molecules encoded by genes whose 1 and 2 coding exons were interchanged are not recognized by T lymphocytes specific for parental class I products. Along with the loss of T -cell reactivity, there is a loss of recognition by some, but not all monoclonal antibodies. In this communication we report that the loss of reactivity by monoclonal antibodies is accompanied by the gain of new epitopes caused by the interaction of 1 and 2 domains. These epitopes are immunodominant. They are the major determinant recognized by polyclonal antisera raised by immunization with L cells transfected with exon-shuffled class I genes. Four new monoclonal antibodies have been produced which recognize at least two separate epitopes caused by the interaction of the 1^P and 2^d domains.     

Mapping the α-globin genes in HB J Mexico carriers

Summary the organization of the -globin genes was studied by restriction endonuclease mapping, in subjects carrying the variant Hb J Mexico. A subject homozygous for Hb J synthesized both Hb J (about 55%) and Hb A and had two loci per chromosome. His restriction site map was found to be identical to that obtained with a normal DNA, except for a mutant Bgl II site which was observed on the Hb J chromosome proximal to the 5-locus. We have also mapped the DNA of a compound heterozygote for Hb J and -thalassemia, who synthesizes 38% Hb J and we have found a single gene corresponding to a –^3.7 haplotype on one chromosome and two genes, respectively ^J and ^A, on the other.     

Frequency and molecular types of deletional α-thalassemia in Egypt

Summary The frequency of deletional -thalassemia in the Egyptian population was estimated at 0.08 by DNA analysis of a newborn random sample. No ⁰ determinants were found. The most frequent ⁺ determinant was the –^3.7 type I in association with the medium allele at inter-zeta HVR. The –^4.2 and ^{anti 3.7} arrangements were found at very low frequencies.     

Modulation of natural killer activity by thymosin alpha 1 and interferon

Summary A single injection of -interferon (-IFN) (30 000 units/mouse), a major biological modifier of natural killer (NK) cytolytic activity, strongly stimulated NK activity in normal mice, as expected, while the same treatment did not statistically alter the NK response in cyclophosphamide (CY)-suppressed animals.We investigated the possibility of thymosin ₁ cooperating with -IFN in boosting NK activity in CY-suppressed animals.The results show that treatment with thymosin ₁ (200 g/kg) for 4 days, followed by a single injection of -IFN 24 h before testing, strongly restored NK activity in CY-suppressed mice. Thymosin ₁ was, moreover, able to accelerate the recovery rate of NK activity in bone marrow reconstituted murine chimeras.Taken together the data support the concept that the synergic effect between thymosin ₁ and -IFN could be the result of effects on differentiation of the NK lineage at different levels.     

Intensity modulated HSQC and HMQC: Two simple methods to measure 3JHNH α in proteins

Two methods for the measurement of homonuclear ³J_HNH coupling constants are described. Both HSQC- and HMQC-type experiments employ `quantitative J-correlation', in which the coupling constant of interest is obtained from the intensity ratio of cross peaks of two spectra. The first spectrum is acquired with ³J_HNH evolution and the second with -proton decoupling. The resolution of these methods in the F₁-domain is not restricted.     

Primer extension studies on α-amylase mRNAs in barley aleurone. II. Hormonal regulation of expression

Relative levels of different -amylase mRNAs were assessed by primer extension experiments using RNA prepared from aleurone of barley (Hordeum vulgare L. cv. Himalaya). Three different aleurone systems were studied: protoplasts prepared from aleurone layers, isolated aleurone layers, and aleurone from germinated grain. Oligonucleotide primers specific for the low-pI and high-pI -amylase groups allowed the levels of different -amylase mRNAs to be assessed both within and between the two groups.In all aleurone systems the same set of -amylase mRNAs was produced in response to either applied gibberellic acid (aleurone protoplasts, isolated aleurone layers) or, presumably, native gibberellin(s) (germinated grain). This result indicates that the same set of genes is being expressed in each case. Differences were observed between the different aleurone systems in regulation of levels of -amylase mRNAs. In particular, the regulation of -amylase mRNA levels in aleurone of germinated grain has unique features which are not adequately explained by the response of isolated aleurone layers to gibberellic acid.     

Effect of α-amanitine on puffing and intranuclear RNA synthesis in Chironomus salivary glands

Incubation of Chironomus salivary glands with -amanitine in concentrations from 1 to 10 /ml results in the suppression of puffing and chromosomal ³H-uridine incorporation after 30 to 60 min in 80% of the cells. Nucleolar ³H-uridine incorporation remains completely unaffected. Even 4 h after the injection of high doses of -amanitine into living larvae, nucleolar incorporation is still pronounced. The distribution of resistant cells within the salivary glands suggests that the uptake of -amanitine is subject to physiological restrictions.—A puff typically induced during in vitro incubation of salivary glands was found to be less sensitive to -amanitine than the Balbiani rings.     

Selection and analysis of non-interactive mutants in the Escherichia coli tryptophan synthase alpha subunit.

Summary The inherent infidelity of Taq DNA polymerase in the polymerase chain reaction was exploited to produce random mutations in thetrp A gene. Screening of the resulting clones allowed selection of non-interactive mutant subunits retaining their intrinsic catalytic activity. Two single changes responsible for this phenotype were identified by DNA sequencing as: 126 valine (GTG)glutamic acid (GAG) and 128 valine (GTT)aspartic acid (GAT). Three single changes giving a non-interactive phenotype with an impaired intrinsic catalytic activity were identified by DNA sequencing as a66 asparagine (AAC)aspartic acid (GAC); 109lysine (AAA) arginine (AGA); 118 cysteine (TGC)arginine (CGC). Where possible, we individually assessed the importance of these residues in interaction in light of structural information from X-ray crystallography and by intergeneric protein sequence comparison.     

Thyroid hormone regulation of Na,K-ATPase subunit-mRNA expression in neonatal rat myocardium

Summary Regulation of Na,K-ATPase mRNA isoform and mRNA expression by thyroid hormone (T₃) in neonatal rat myocardium was examined. In euthyroid neonates between ages of 2 and 5 days, mRNA₁, mRNA₃, and mRNA₁ abundances were nearly constant while mRNA₂ was undetectable. During the interval between postnatal days 5 and 15, mRNA₃ decreased to negligible levels and mRNA₂ became expressed and increased in abundance to account for 20% of the mRNA pool by the 15^th postnatal day. To examine the effect of T₃ on this developmental program, neonates were injected with 75 g T₃/100 g body weight or diluent alone on the second and third postnatal days and myocardial Na,K-ATPase subunit-mRNA abundances were determined on the third and fourth postnatal days. Because T₃ treatment increased the RNA/DNA ratios of myocardial tissue, the subunit-mRNA abundances were normalized per unit DNA. Following 24 and 48 hr of T₃ treatment, the abundances of mRNA₁, mRNA₃, and mRNA₁ increased, while mRNA₂ continued to remain undetectable during the 2-day interval between the second to fourth postnatal days. It is concluded that T₃ augments the abundance of Na,K-ATPase subunit mRNAs that are already being expressed in the neonatal rat myocardium. The results further suggest that T₃ does not act as a molecular switch in the developmental expression of the mRNA isoforms in rat myocardium during the first four postnatal days.     

Measurement of the protein backbone dihedral angle φ based on quantification of remote CSA/DD interference in inter-residue 13C′(i − 1)-13Cα(i) multiple-quantum coherences

A novel triple-resonance NMR method is presented for the measurement of the protein backbone dihedral angle based on differential multiple-quantum relaxation induced by relaxation interference between ¹H(i)-¹³C(i) dipolar and ¹³C(i–1) (carbonyl) chemical shift anisotropy mechanisms. The method employs a simultaneous transfer of ¹⁵N magnetization to the inter- and intra-residue ¹³C carbons as well as the directly attached carbonyl carbon ¹³C. Results obtained on ¹³C,¹⁵N-labeled ubiquitin demonstrate the potential of the method.