Essential oil composition of two yarrow taxonomic forms

Differences in essential oil composition of wild Achillea millefolium L., collected at five habitats in Lithuania, where plants with pink (f. rosea) and white (f. millefolium) flowers grow together, were reported. For the first time, oils of different plant organs (inflorescences and leaves) of both forms from every population were analysed in detail. Chemical analysis was performed by GC and GC-MS. The most predominant constituents of the oils were nerolidol (9.4–31.9%, in 11 out of 20 samples), caryophyllene oxide (8.4–23.0%, 4 leaf oils), ß-pinene (8.0–15.2%, 2 samples), eudesmol (11.8–15.8%, 2 leaf oils) and 1,8-cineole (11.9%, one inflorescence oil). Domination of nerolidol was mostly characteristic of A. millefolium f. rosea essential oils (in 8 out of 10 oils). The inflorescences biosynthesised markedly larger amounts of nerolidol and ß-pinene than those of the leaves. An opposite correlation was observed for caryophyllene oxide and eudesmol. Chamazulene (≤2.7%) was determined only in six oils. The 65 identified constituents made up 75.4–96.5% of the oils.     

Tightly bound to DNA proteins: possible universal substrates for intranuclear processes

Tightly bound to DNA proteins (TBPs) are a protein group that remains attached to DNA after its deproteinization by phenol, chloroform or salting-out. TBP are bound to DNA with covalent phosphotriester or non-covalent ion and hydrogen bonds. They appear to be a vast protein group involved in numerous intranuclear processes. The TBPs fraction co-purified with DNA deproteinized by mild procedures is extremely heterogeneous, tissue and species-specific. The protein fraction co-purified with DNA after harsh deproteinization procedures appears to be formed from few polypeptides common to different species and tissues. Interaction sites between DNA and TBPs depend on the physiological status of the cell. The binding sites of TBPs to DNA do not co-localize with the nuclear matrix attachment regions. We hypothesize that TBPs form a universal substrate for intranuclear processes.     

Development-dependent changes in the tight DNA-protein complexes of barley on chromosome and gene level

Background  

The tightly bound to DNA proteins (TBPs) is a protein group that remains attached to DNA with covalent or non-covalent bonds after its deproteinisation. The functional role of this group is as yet not completely understood. The main goal of this study was to evaluate tissue specific changes in the TBP distribution in barley genes and chromosomes in different phases of shoot and seed development. We have: 1. investigated the TBP distribution along Amy32b and Bmy1 genes encoding low pI α-amylase A and endosperm specific β-amylase correspondingly using oligonucleotide DNA arrays; 2. characterized the polypeptide spectrum of TBP and proteins with affinity to TBP-associated DNA; 3. localized the distribution of DNA complexes with TBP (TBP-DNA) on barley 1H and 7H chromosomes using mapped markers; 4. compared the chromosomal distribution of TBP-DNA complexes to the distribution of the nuclear matrix attachment sites.     

Distribution of tight DNA-protein complexes along the barley chromosome 1H, as revealed by microsatellite marker analysis

The distribution of DNA complexes with proteins resistant to routine deproteinisation procedures (tightly bound proteins, TBP) was studied on the barley chromosome 1H by means of microsatellite analysis. The polypeptide spectrum of the barley shoot TBP was similar to that formerly described for other organisms. In order to reveal developmental changes in the distribution of the TBP, DNA was extracted from dry grains, coleoptiles, root tips, and young and old leaves. In the seeds, all the studied DNA sites were evenly distributed between free DNA and DNA containing the tight DNA-protein complexes. Germination made the interaction between TBP and chromosomal loci specific. In coleoptile DNA, sites containing microsatellites located in the distal part of the long arm of the chromosome were not bound to the TBP anymore, however, the centromeric markers were found exclusively in the tight DNA-protein complexes. A similar but not identical distribution of markers was observed in the root tips and young leaves. Leaf senescence was accompanied by a loss in interaction specificity between chromosomal loci and tightly bound proteins. These results are considered to reflect changes in chromatin domain interaction with the nuclear matrix during plant development.     

A new rapid method for quantitating radioactive proline, 4-hydroxyproline,and 3-hydroxyproline

A simple and rapid method for separating proline, 4-hydroxyproline and 3-hydroxyproline was developed by the use of high-voltage electrophoresis. An excellent counting efficiency of proline was achieved by a simple extraction of the labeled material from paper; 98–100% recovery of count rates was obtained, equivalent to recoveries from column chromatography. This method can tolerate high concentrations of salt, acid, and protein in the sample. No carrier is required and multiple samples (up to 15) can be separated on a single sheet and analyzed within 2 h. Serial dilution experiments showed excellent linearity. An average recovery rate of 92% was obtained for samples over a wide range of radioactivity and high sensitivity of the method was demonstrated. This analysis is applicable to protein hydrolysates and to determination of the free amino acids in the presence of protein. Thus, proline, 4-hydroxyproline and 3-hydroxyproline can be quantitated simultaneously in any biological sample.     

Macrophage-T cell interaction mediated by immunogenic and non-immunogenic forms of a monofunctional antigen

Summary As an approach to the elucidation of the essential steps in the immune pathway, the uptake and retention of immunogenic and non-immunogenic analogs of a monofunctional antigen by guinea pig macrophages and the efficiency of macrophages pulsed with the compounds to present antigen to sensitized T lymphocytes were compared. L-Tyrosine-azobenzene-p-arsonate (RAT) and its non-immunogenic analog, 4-hydroxyphenyl-n-propane-3-azobenzene-p-arsonate (RAN), react similarly with antiarsonate antibody, but RAN, unlike RAT, is unable to induce cellular immunity in guinea pigs. The uptake and retention patterns of the two compounds by macrophages differed in that, at a given time, more RAN than RAT was retained and detectable on cell surfaces by anti-arsonate antibody. Equivalent numbers of T lymphocytes from guinea pigs sensitized to RAT formed antigen-dependent clusters with macrophages pulsed with either RAT or RAN after 24 hr in culture, but not with macrophages pulsed with an azobenzenoid compound of unrelated specificity. On the other hand, T lymphocytes from guinea pigs immunized with RAN showed no significant capacity to bind to macrophages which had been pulsed with any of the compounds. The number of lymphocytes from RAT-sensitized animals which bound to RAT-pulsed macrophages remained relatively stable over a 48 hr period, whereas clusters of the same lymphocytes with RAN-pulsed macrophages dissocitated to background levels within that time. Early cluster formation mediated by RAN, as well as its ability to induce transient specific T cell unresponsiveness to RAT in vivo, indicate that T cells are capable of recognizing (binding) the non-immunogen. However, such early, and perhaps weak, interaction with RAN-pulsed macrophages did not induce DNA synthesis by T cells. Anti-Ia serum completely blocked cluster formation mediated by either RAT or RAN. Thus, the only significant distinction disclosed by these studies between the immunogenic and non-immunogenic compounds was the stability of macrophage-T cell interaction as determined by the persistence of antigen mediated cell clusters in culture, suggesting that this may be a factor in immunogenic discrimination.Abbreviations ABA azobenzenearsonate - BSA bovine serum albumin - CFA complete Freund's adjuvant - IFA incomplete Freund's adjuvant - KLH keyhole limpet hemocyanin - LNC Lymph node cells - MHC major histocompatibility complex - PEC peritoneal exudate cells - PEL peritoneal exudate lymphocytes - RAN 4-hydroxyphenyl-n-propane-3-azobenzene-p-arsonate - RAT L-tyrosine-azobenzene-p-arsonate - TAT L-tyrosine-azobenzene-p-trimethylammonium chlorideAided by USPHS Grant AI 05664.     

Related bifunctional restriction endonuclease-methyltransferase triplets: TspDTI, Tth111II/TthHB27I and TsoI with distinct specificities

Background

We previously defined a family of restriction endonucleases (REases) from Thermus sp., which share common biochemical and biophysical features, such as the fusion of both the nuclease and methyltransferase (MTase) activities in a single polypeptide, cleavage at a distance from the recognition site, large molecular size, modulation of activity by S-adenosylmethionine (SAM), and incomplete cleavage of the substrate DNA. Members include related thermophilic REases with five distinct specificities: TspGWI, TaqII, Tth111II/TthHB27I, TspDTI and TsoI.

Results

TspDTI, TsoI and isoschizomers Tth111II/TthHB27I recognize different, but related sequences: 5'-ATGAA-3', 5'-TARCCA-3' and 5'-CAARCA-3' respectively. Their amino acid sequences are similar, which is unusual among REases of different specificity. To gain insight into this group of REases, TspDTI, the prototype member of the Thermus sp. enzyme family, was cloned and characterized using a recently developed method for partially cleaving REases.

Conclusions

TspDTI, TsoI and isoschizomers Tth111II/TthHB27I are closely related bifunctional enzymes. They comprise a tandem arrangement of Type I-like domains, like other Type IIC enzymes (those with a fusion of a REase and MTase domains), e.g. TspGWI, TaqII and MmeI, but their sequences are only remotely similar to these previously characterized enzymes. The characterization of TspDTI, a prototype member of this group, extends our understanding of sequence-function relationships among multifunctional restriction-modification enzymes.