Conformation and formation tendency of the cyclotetrapeptide cyclo (D-Pro-D-Pro-L-Pro-L-Pro): experimental results and molecular modeling studies

The title compound represents the smallest member of cyclic proline peptides corresponding to the general formula c(DDLL-Pro4)n with a strictly D,D,L,L double-alternating sequence of the chiral amino acid residues. The cyclopeptides with n greater than or equal to 2 could be synthesized from both DDLL-Pro4 (1) and DLLD-Pro4 (2). The cyclic monomer (n = 1) resulted only from 2, whereas not even a trace could be found by cyclization of 1. The peptide exists in a strongly strained Ci symmetrical conformation (x-ray analysis) with alternating cis and trans peptide bonds (ctct form I). The cis peptide bonds deviate from planarity (omega = 22 degrees); two of the pyrrolidine rings show a "South" conformation (phi = -94 degrees), whereas the other residues exhibit C alpha-endo puckering (phi = -124 degrees). Two of the psi angles surprisingly occur at +41 degrees (anti-cis'), the others are located in the trans' region. A quantitative ring opening occurs with trifluoroacetic acid at room temperature. In solution the existence of an isomeric ctcc sequence (form Ia) is indicated. Dreiding model studies also suggested a favorable conformation with a tctc sequence (form II). Consequently, we performed molecular mechanics calculations, based on the CHARMM force field and semiempirical quantum mechanical AM1 calculations (MOPAC program). Pronounced differences in the backbone parameters were found using these two methods. However, the theoretical studies evidenced the experimentally obtained differences in the cyclization tendencies of the linear precursors.     

Transformed 3T3 cells have reduced levels and altered subcellular distribution of the major PKC substrate protein marcks

The MARCKS (myristylated alanine-rich C-kinase substrate) protein is an abundant calmodulin-binding protein that is a major and specific endogenous substrate of protein kinase C (PKC). Stimulation of cells with phorbol esters or other activators of PKC has been shown previously to result in rapid phosphorylation of MARCKS proteins and redistribution of these myristylated C-kinase substrates from membrane to cytosol. Here we show that NIH3T3 murine fibroblasts transformed by p21-HA-C-RAS or pp60-V-SRC oncoproteins have markedly reduced levels of p68-MARCKS and that most of the remaining MARCKS protein is found in the cytosol. 3T3 cells containing a nontransforming oncoprotein p26-BCL2, in contrast, exhibited normal levels and distribution of p68-MARCKS. When taken together with recent evidence that MARCKS proteins are involved in regulating organization of the membrane cytoskeleton, our findings suggest that oncoprotein-mediated alterations in MARCKS protein levels and subcellular distribution may contribute to the development or maintenance of the transformed phenotpe.     

A common sequence in the inverted terminal repetitions of human and avian adenoviruses

The termini of the avian chick embryo lethal orphan (CELO) virus DNA have been sequenced. The results revealed a 63-bp-long inverted terminal repetition (ITR) which shared the sequence ATAATA with all adenovirus termini, thus far analyzed. The CELO virus ITR differed from those of the mammalian adenoviruses in two major aspects: (i) it is not a perfect duplication; (ii) it begins with a 5'-guanylic acid residue instead of the cytidylic acid normally observed in adenoviruses.     

Specific labelling of the active site of the phosphate translocator in spinach chloroplasts by 2,4,6-trinitrobenzene sulfonate

Phosphate transport across the chloroplast envelope is rapidly inactivated by the amino-group reagent 2,4,6-trinitrobenzene sulfonate. Subsequent exposure to [³H]NaBH₄ leads to an incorporation of the trinitrophenyl moiety into envelope membrane preparations. From the membrane proteins only a polypeptide with 29000 dalton molecular weight is labelled. The inactivation of phosphate transport and the incorporation of radioactivity are both specifically reduced by the presence of substrates.The results lead to the conclusion that a polypeptide with a molecular weight of 29000 dalton and containing a lysyl residue at the substrate binding site is involved in the phosphate translocator function.     

Alterations in the lipid content of rat palatal epithelium during carcinogenesis

Summary The hard palates of 150 female albino rats of the Sprague-Dawley strain were painted 3 times a week either with the fat-soluble carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) for 11 months with inhibited secretion of saliva or with the water-soluble carcinogen 4-nitrochinoline N-oxide (4NQO) for 8 months with intact salivary secretion. Specimens were taken regularly from the mucosa of the hard palate, and the content of lipids in the epithelium was studied histochemically and biochemically during the carcinogenesis.Changes in the lipid content could be observed histochemically as there was a focal loss of lipid stainability in the epithelium during the more advanced stages of carcinogenesis with severe epithelial dysplasia, carcinoma in situ and early invasive carcinoma.The biochemical method used (TLC) could not, however, verify the histologically observed changes in the lipid content of the epithelium, probably because the changes were very local.The investigations have been performed with financial support from Maggie Stephens Foundation, Colgate-Palmolive Limited and from the Swedish Dental Society.     

Coiled-Coil Formation on Lipid Bilayers—Implications for Docking and?Fusion Efficiency

Coiled-coil formation of four different oligopeptides was characterized in solution, on hydrogels, and on membranes by employing circular dichroism spectroscopy, surface plasmon resonance spectroscopy, attenuated total reflection infrared spectroscopy, and ellipsometry. Peptide sequences rich in either glutamic acid (E: E3Cys, i-E3Cys) or lysine (K: K3Cys, i-K3Cys) were used to represent minimal mimics of eukaryotic SNARE motifs. Half of the peptides were synthesized in reverse sequence, so that parallel and antiparallel heptad coiled-coil structures were formed. Either E-peptides or K-peptides were attached covalently to phospholipid anchors via maleimide chemistry, and served as receptors for the recognition of the corresponding binding partners added to solution. Attenuated total reflection infrared spectroscopy of single bilayers confirmed the formation of coiled-coil complexes at the membrane interface. Coiled-coil formation in solution, as compared with association at the membrane surface, displays considerably larger binding constants that are largely attributed to loss of translational entropy at the interface. Finally, the fusogenicity of the various coiled-coil motifs was explored, and the results provide clear evidence that hemifusion followed by full fusion requires a parallel orientation of α-helices, whereas antiparallel oriented coiled-coil motifs display only docking.     

GROWTH AND ORGANIC OSMOLYTES OF GEOGRAPHICALLY DIFFERENT ISOLATES OF MICROCOLEUS CHTHONOPLASTES (CYANOBACTERIA) FROM BENTHIC MICROBIAL MATS:RESPONSE TO SALINITY CHANGE1

The comparative growth and osmotic acclimation often culture strains of the marine benthic cyanobacterium Microcoleus chthonoplastes Thuret isolated from microbial mats in Germany, Spain, Egypt, the United States, Mexico, Chile, and Australia were investigated in salinities ranging from freshwater to twice seawater. All isolates showed a broad growth versus salinity response consistent with the dominance of this species in intertidal and hypersaline microbial communities. Growth optima, salinity preferences, and maximum growth rates differed for each isolate and could be related to the habitat from which they were isolated. This is most obvious when comparing strains from brackish habitats with those from a hypersaline lake. While the former isolates exhibited sharply pronounced growth optima under hyposaline conditions, cultures from the hypersaline environment grew best in salinity more than double seawater. The major low-molecular weight organic compounds present in all M. chthonoplastes strains were the carbohydrates glycosylglycerol and trehalose. This was proven by using ¹³C-nuclear magnetic resonance spectroscopy. Glycosylglycerol was synthesized and accumulated with increasing salinities, indicating its role as an osmolyte. In contrast, trehalose was present in relatively high concentrations under hyposaline conditions only. Differences in the patterns of growth versus salinity, as well as in those of osmotic acclimation among the M. chthonoplastes isolates, point to the development of different physiological ecotypes within the species.     

Desert breath—How fog promotes a novel type of soil biocenosis,forming the coastal Atacama Desert’s living skin

The Atacama Desert is the driest non‐polar desert on Earth, presenting precarious conditions for biological activity. In the arid coastal belt, life is restricted to areas with fog events that cause almost daily wet–dry cycles. In such an area, we discovered a hitherto unknown and unique ground covering biocenosis dominated by lichens, fungi, and algae attached to grit‐sized (~6 mm) quartz and granitoid stones. Comparable biocenosis forming a kind of a layer on top of soil and rock surfaces in general is summarized as cryptogamic ground covers (CGC) in literature. In contrast to known CGC from arid environments to which frequent cyclic wetting events are lethal, in the Atacama Desert every fog event is answered by photosynthetic activity of the soil community and thus considered as the desert's breath. Photosynthesis of the new CGC type is activated by the lowest amount of water known for such a community worldwide thus enabling the unique biocenosis to fulfill a variety of ecosystem services. In a considerable portion of the coastal Atacama Desert, it protects the soil from sporadically occurring splash erosion and contributes to the accumulation of soil carbon and nitrogen as well as soil formation through bio‐weathering. The structure and function of the new CGC type are discussed, and we suggest the name grit–crust. We conclude that this type of CGC can be expected in all non‐polar fog deserts of the world and may resemble the cryptogam communities that shaped ancient Earth. It may thus represent a relevant player in current and ancient biogeochemical cycling.