What is the real crystallographic structure of the L photointermediate of bacteriorhodopsin?

In the last few years, three laboratories have reported three entirely different crystallographic models for the L photointermediate of bacteriorhodopsin. All are from X-ray diffraction of illuminated crystals that contain L in photostationary states created at similar cryogenic temperatures. This article compares the models and their implications, the crystallographic statistics and the methods used to derive them, as well as their agreement with non-crystallographic information.     

Suppression of Lα/Lβ Phase Coexistence in the Lipids of Pulmonary Surfactant

To determine how different constituents of pulmonary surfactant affect its phase behavior, we measured wide-angle x-ray scattering (WAXS) from oriented bilayers. Samples contained the nonpolar and phospholipids (N&PL) obtained from calf lung surfactant extract (CLSE), which also contains the hydrophobic surfactant proteins SP-B and SP-C. Mixtures with different ratios of N&PL and CLSE provided the same set of lipids with different amounts of the proteins. At 37°C, N&PL by itself forms coexisting L_α and L_β phases. In the L_β structure, the acyl chains of the phospholipids occupy an ordered array that has melted by 40°C. This behavior suggests that the L_β composition is dominated by dipalmitoyl phosphatidylcholine (DPPC), which is the most prevalent component of CLSE. The L_β chains, however, lack the tilt of the L_β′ phase formed by pure DPPC. At 40°C, WAXS also detects an additional diffracted intensity, the location of which suggests a correlation among the phospholipid headgroups. The mixed samples of N&PL with CLSE show that increasing amounts of the proteins disrupt both the L_β phase and the headgroup correlation. With physiological levels of the proteins in CLSE, both types of order are absent. These results with bilayers at physiological temperatures indicate that the hydrophobic surfactant proteins disrupt the ordered structures that have long been considered essential for the ability of pulmonary surfactant to sustain low surface tensions. They agree with prior fluorescence micrographic results from monomolecular films of CLSE, suggesting that at physiological temperatures, any ordered phase is likely to be absent or occupy a minimal interfacial area.     

Modulation of the orientational order profile of the lipid acyl chain in the Lα phase

The orientational order profile along the lipid acyl chain has been characterized under several different conditions of polar headgroup composition, temperature, and cholesterol content. Despite the different nature of these factors, the variation of the order is governed by two common trends. First, the relative change of order induced by the variation of these factors is always more pronounced towards the end of the chain than for the methylene groups near the interface. Second, there is, to a first approximation, a distinct correlation between the magnitude of the order parameters and the shape of the order profile. For example when the chain is highly ordered, the relative width of the order distribution is narrow indicating that the plateau region is longer. These conclusions suggest that the orientational order profile depends on only a small number of parameters and demonstrate clearly that the correlation length for changes in orientational order is much greater than one C-C bond length. Our results also show that the reduced temperature is not related in simple terms to orientational order and probably has little theoretical significance. The orientational order profiles of POPC and POPE bilayers are significantly different even when expressed in terms of reduced temperature. The behavior of POPC/cholesterol systems also indicates that the orientational order of the lipid chain and the gel-to-liquid crystalline phase transition temperature are not related in a straightforward manner.Abbreviations POPC 1-palmitoyl-2-oleoyl-phosphatidylcholine - POPE 1-palmitoyl-2-oleoyl-phosphatidylethanolamine - PC phosphatidylcholine - PE phosphatidylethanolamine - NMR nuclear magnetic resonance - EDTA ethylenediaminetetraacetic acid Offprint requests to: M. Bloom     

The formation of vesicles in the developmental cycle of the nodular endophyte of Hippophaë rhamnoides L.

Summary The mature vesicle of the Hippophaë rhamnoides root nodule endophyte is spherical, approximately 3–4 m in diameter and exhibits a high degree of apparently random septation. Electron micrographs are presented which show that this vesicular form in the endophytic development within the host cell originates as a swelling of the hyphal tip. The young vesicle is non-septate and in general attains a minimum diameter of 3 m before septation becomes evident. The number of septa then increases with vesicle maturity.     

Parasite fauna of the burbot Lota lota L. in body of water of the Kol'skiĭ peninsula

The results of a parasitological study of the burbot Lota Iota L. inhabiting the Kola region are presented. 51 species of parasite were found on burbot in 16 waterbodies belonging to the White Sea and Barents Sea basins (Muxosporea - 7, Suctoria - 1, Peritricha - 6, Monogenea - 1, Cestoda - 6, Trematoda - 13, Nematoda - 6, Acanthocephala - 5, Hirudinea - 3, Bivalvia - 1 and Crustacea - 2 species). Data on the infestation of burbot by different parasite species and their prevalence in investigated waterbodies were obtained.     

Observations on the fine structure of the root nodule endophyte of Hippophaë rhamnoides L.

Summary Electron microscopy of ultra-thin sections of Hippophaë rhamnoides root nodules has been carried out in order to elucidate the nature of the endophyte. The organism is seen as a branching, septate filament approximately 0.6 microns in diameter bearing on its terminal ends spherical sub-divided vesicles 3–4 microns in diameter. In the mature nodule the vesicles are the most prominent endophyte form and appear to be formed by swelling of the hyphal tips. It is concluded that the endophyte is an actinomycete closely related to but not identical with that of Alnus glutinosa.     

The Role of Dosage of the Region 7d1–7d5-6 of the X Chromosome in the Production of Homeotic Transformations in DROSOPHILA MELANOGASTER

A high frequency of homeotic transformations appears in Df(3)red/+ progeny of Df(1)sn^C128 /+ females. Generally, the metathoracic appendages are partially transformed into mesothoracic ones. Df(1)sn^C128 includes a small region of the X chromosome: 7D1 to 7D5-6. Hypodosage of this region is mainly effective at the level of the maternal genotype, and the effect is probably due to hypodosage of the wild-type allele of the gene fs(1)h. Df(3)red has an effect that is mainly, if not exclusively, zygotic, probably due to hypodosage of the wild-type allele of Rg-bx. The frequencies of transformed flies resulting from the interaction between Df(1)sn^C128 and Df(3)red are not very sensitive to external conditions and genetic background. Studies of the interactions between Df(1)sn^C128 and other mutations or deficiencies of chromosome 3 [Rg-pbx, bx, pbx, Ubx¹, Ubx¹³⁰, Ubx⁸⁰, Df(3)P9] reveal an analogy between the hypodosage effect of region 7D1–7D5-6 and the effects of ether treatment of blastoderm stage eggs. The role of the gene fs(1)h in the process of segment determination is discussed in the light of these results.     

Studies on the Constituents of Typha L.—IV. A Preliminary Study of Chemosystematics of Typha L.

From pollen grains of Typha davidiana, T. latifolia, T. angustata the same eight flavonoids have been isolated. They are identified as naringenin I, isorhamnetin II, quercetin III, isorhamnetin-3-O-(2^G-α-L-rhamnopyranosyl)-rutioside IV, quercetin-3-O-(2^G-α-L-rhamnopyranosyl)-rutinosida, V, isorhamnetio-3-O-rutinoside VI, isorhamnetino-3-O-neohesperidoside VII, kampferol-3-O-neohesperidoside VIII. Flavonoids of pollen grains of five species of Typha, including the above three species, were analysed by TLC with the result showing that the constituents in the pollen grains of the five species are very similar. The chemical comparison among Typha and Sparganium and 16 possibly related families shows that Typha is different from Pandanaceae or Pandanales and is similar to Restionaceae, Flagellariaceae, Juncaceae and Cyperaceae in some respects. Typha and Sparganium are very similar in many respects, and they could be treated in the same family, Typhaceae, which merit the rank of order, Typhales.     

Nicotinic Acetylcholine Receptors Containing the α7-Like Subunit Mediate Contractions of Muscles Responsible for Space Positioning of the Snail,Helix pomatia L. Tentacle

Three recently discovered tentacle muscles are crucial to perform patterned movements of upper tentacles of the terrestrial snail, Helix pomatia. The muscles receive central and peripheral excitatory cholinergic innervation lacking inhibitory innervation. Here, we investigate the pharmacology of acetylcholine (ACh) responses in muscles to determine the properties of the ACh receptor (AChR), the functional availability of which was assessed using isotonic contraction measurement. Using broad spectrum of nicotinic and muscarinic ligands, we provide the evidence that contractions in the muscles are attributable to the activation of nAChRs that contain the α7-like subunit. Contractions could be evoked by nicotine, carbachol, succinylchloride, TMA, the selective α7-nAChR agonist choline chloride, 3-Bromocytisine and PNU-282987, and blocked by nAChR selective antagonists such as mytolon, hexamethonium, succinylchloride, d-tubocurarine, hemicholinium, DMDA (decamethonium), methyllycaconitine, α-Bungarotoxin (αBgTx) and α-Conotoxin IMI. The specific muscarinic agonist oxotremorine and arecoline failed to elicit contractions. Based on these pharmacological properties we conclude that the Na⁺ and Ca²⁺ permeable AChRs of the flexor muscle are nicotinic receptors that contain the α7-like subunit. Immunodetection experiments confirmed the presence of α7- or α7-like AChRs in muscle cells, and α4-AChRs in nerves innervating the muscle. These results support the conclusion that the slowly desensitizing αBgTx-sensitive responses obtained from flexor muscles are produced by activation of α7- like AChRs. This is the first demonstration of postsynaptic expression and an obligatory role for a functional α7-like nAChR in the molluscan periphery.     

Regulation of α-galactosidase activity and the hydrolysis of galactomannan in the endosperm of the fenugreek (Trigonella foenum-graecum L.) seed

When endosperms were isolated from fenugreek seeds 5 h after sowing and incubated in a small volume of water, the development of α-galactosidase activity and the breakdown of the galactomannan storage polysaccharide were both inhibited relative to control endosperms incubated in larger volumes. The inhibition could be relieved by pre-washing the endosperms, and reimposed by the wash-liquors. If the endosperms were isolated 24 h after sowing, no inhibition was observed. Removal of the embryonic axis from germinating fenugreek seeds and from germinated seedlings also inhibited the development of α-galactosidase activity and galactomannan breakdown in the endosperms; the inhibition was more pronounced the earlier the axis was removed. Axis excision 5 h after sowing caused a delay in the onset of galactomannan breakdown and of the appearance of α-galactosidase activity in the endosperms. It also led to a decrease in the rates of galactomannan breakdown and α-galactosidase production. Axis excision 24 h after sowing caused only a slowing of the rates of galactomannan breakdown and α-galactosidase increase. The inhibition caused by axis removal at 5 h could be relieved partially by gibberellin (10^-4 M), benzyladenine (10^-5 M), mixtures of these and by the herbicide SAN 9789 [4-chloro-5-(methylamine)-2-(α,α,α-trifluoro-m-tolyl)-3-(2H)-pyridazinone]. These substances had no effect on the inhibition caused by axis-removal at 24 h. Excision of the cotyledons at 5 h-leaving the separated axis and the endosperm-also caused inhibition of galactomannan breakdown and α-galactosidase development. The results are consistent with the presence in the fenugreek seed endosperm of diffusible inhibitors of galactomannan mobilisation which are removed or inactivated during normal germination and early seedling development. They are also consistent with a role for the seedling axis in the control of galactomannan breakdown in the endosperm. Initially the axis appears to have a regulatory function (via gibberellins and/or cytokinins?) in determining the onset of α-galactosidase production in the endosperm. Thereafter its continued presence is necessary to ensure maximal rates of α-galactosidase production and galactomannan hydrolysis. The role of the axis may be initially to counteract the endogenous inhibitors in the endosperm and then to act as a sink for the galactomannan breakdown products released in the endosperm and taken up by the cotyledons.     

Organization of the interferon-inducible 2′,5′-oligoadenylate-dependent ribonuclease L (RNase L) gene of mouse

Interferons (IFNs) induce a 2′,5′-oligoadenylate (2-5A)-dependent ribonuclease L (RNase L) following virus-infection of mammalian cells. RNase L degrades both viral and cellular RNAs and restricts virus-proliferation. We have studied organization of RNase L gene in genomic DNA from the mouse liver by Southern blot analysis. Several BamHI, BglII, EcoRI, HincII, HindIII, NcoI, PstI, SacI, and XbaI restriction fragments hybridized to ³²P-labeled mouse RNase L cDNA and the 5′-proximal exon probes. Mouse RNase L gene exists as a single copy (>16 kb DNA) gene. A 5 kb HindIII and a 2.5 kb EcoRI DNA were detected as 5′-upstream DNA of the gene which may contain mouse RNase L promoter. Our results will help studying mouse RNase L gene promoter in further details.     

Glycine origin of the methyl substituent on C7′-N of octodiose for the biosynthesis of apramycin

Apramycin is unique in the aminoglycoside family due to its octodiose moiety. However, either the biosynthesis process or the precursors involved are largely unknown. Addition of glycine, as well as serine or threonine, to the Streptomyces tenebrabrius UD2 fermentation medium substantially increases the production of apramycin with little effect on the growth of mycelia, indicat-ing that glycine and/or serine might be involved in the biosynthesis of apramycin. The ¹³C-NMR analysis of [2-¹³C] glycine-fed (25% enrichment) apramycin showed that glycine specifically and efficiently incorporated into the only N-CH₃ substituent of apramycin on the C7′ of the octodiose moiety. We noticed that the in vivo concentration of S-adenosyl methionine increased in parallel with the addition of glycine, while the addition of methione in the fermentation medium significantly decreased the productivity of apramycin. Therefore, the methyl donor function of glycine is proposed to be involved in the methionine cycle but methionine itself was proposed to inhibit the methylation and methyl transfer processes as previously reported for the case of rapamycin. The ¹⁵N NMR spectra of [2-¹³C,¹⁵N]serine labeled apramycin indicated that serine may also act as a limiting precursor contributing to the ―NH₂ substituents of apramycin.     

Mapping the membrane topography of the TH6–TH7 segment of the diphtheria toxin T-domain channel

Low pH triggers the translocation domain of diphtheria toxin (T-domain), which contains 10 α helices, to insert into a planar lipid bilayer membrane, form a transmembrane channel, and translocate the attached catalytic domain across the membrane. Three T-domain helices, corresponding to TH5, TH8, and TH9 in the aqueous crystal structure, form transmembrane segments in the open-channel state; the amino-terminal region, TH1–TH4, translocates across the membrane to the trans side. Residues near either end of the TH6–TH7 segment are not translocated, remaining on the cis side of the membrane; because the intervening 25-residue sequence is too short to form a transmembrane α-helical hairpin, it was concluded that the TH6–TH7 segment resides at the cis interface. Now we have examined this segment further, using the substituted-cysteine accessibility method. We constructed a series of 18 mutant T-domains with single cysteine residues at positions in TH6–TH7, monitored their channel formation in planar lipid bilayers, and probed for an effect of thiol-specific reagents on the channel conductance. For 10 of the mutants, the reagent caused a change in the single-channel conductance, indicating that the introduced cysteine residue was exposed within the channel lumen. For several of these mutants, we verified that the reactions occurred primarily in the open state, rather than in the flicker-closed state. We also established that blocking of the channel by an amino-terminal hexahistidine tag could protect mutants from reaction. Finally, we compared the reaction rates of reagent added to the cis and trans sides to quantify the residue’s accessibility from either side. This analysis revealed abrupt changes in cis- versus trans-side accessibility, suggesting that the TH6–TH7 segment forms a constriction that occupies a small portion of the total channel length. We also determined that this constriction is located near the middle of the TH8 helix.     

Protein P7 of the Cystovirus φ6 Is Located at the Three-Fold Axis of the Unexpanded Procapsid

The objective of this study was to determine the location of protein P7, the RNA packaging factor, in the procapsid of the φ6 cystovirus. A comparison of cryo-electron microscopy high-resolution single particle reconstructions of the φ6 complete unexpanded procapsid, the protein P2-minus procapsid (P2 is the RNA directed RNA-polymerase), and the P7-minus procapsid, show that prior to RNA packaging the P7 protein is located near the three-fold axis of symmetry. Difference maps highlight the precise position of P7 and demonstrate that in P7-minus particles the P2 proteins are less localized with reduced densities at the three-fold axes. We propose that P7 performs the mechanical function of stabilizing P2 on the inner protein P1 shell which ensures that entering viral single-stranded RNA is replicated.     

The Functional Role of the Hollow Region of the Butterfly Pyrameis atalanta （L.） Scale

Questions concerning the functional role of the hollow region of the butterfly Pyrameis atalanta （L.） scale are experimentally investigated. Attention was initially directed to this problem by observation of the complex microstrucmre of the butterfly scale as well as other studies indicating higher lift on butterfly wings covered with scale. The aerodynamic forces were measured for two oscillating scale models. Results indicated that the air cavity of an oscillating model of the Pyrameis atalanta （L.） scale increased the lift by a factor of 1.15 and reduced the damping coefficients by a factor of 1.38. The modification of the aerodynamic effects on the model of butterfly scale was due to an increase of the virtual air mass, which influenced the body. The hollow region of the scale increased the virtual air mass by a factor of 1.2. The virtual mass of the butterfly scale with the hollow region was represented as the sum of air mass of two imaginary geometrical figures： a circular cylinder around the scale and a right-angled parallelepiped within the hollow region. The interaction mechanism of the butterfly Pyrameis atalanta （L.） scale with a flow was described. This novel interaction mechanism explained most geometrical features of the airpermeable butterfly scale （inverted V-profile of the ridges, nozzle of the tip edge, hollow region, and openings of the upper lamina） and their arrangement.