Compositional heterogeneity reflects partial dehydration in three-dimensional crystals of bacteriorhodopsin

Absorption, fluorescence and excitation spectra of three-dimensional bacteriorhodopsin crystals harvested from a lipidic cubic phase are presented. The combination of the spectroscopic experiments performed at room temperature, controlled pH and full external hydration reveals the presence of three distinct protein species. Besides the well-known form observed in purple membrane, we find two other species with a relative contribution of up to 30%. As the spectra are similar to those of dehydrated or deionized membranes containing bacteriorhodopsin, we suggest that amino acid residues, located in the vicinity of the retinal chromophore, have changed their protonation state. We propose partial dehydration during crystallization and/or room temperature conditions as the main source of this heterogeneity. This assignment is supported by an experiment showing interconversion of the species upon intentional dehydration and by crystallographic data, which have indicated an in-plane unit cell in 3D crystals comparable to that of dehydrated bacteriorhodopsin membranes. Full hydration of the proteins after the water-withdrawing crystallization process is hampered. We suggest that this hindered water diffusion originates mainly from a closure of hydrophobic crystal surfaces by lipid bilayers. The present spectroscopic work complements the crystallographic data, due to its ability to determine quantitatively compositional heterogeneity resulting from proteins in different protonation states.     

Sequence heterogeneity of TT virus and closely related viruses

TT virus (TTV) is a recently discovered infectious agent originally obtained from transfusion-related hepatitis. However, the causative link between the TTV infection and liver disease remains uncertain. Recent studies demonstrated that genome sequences of different TTV strains are significantly divergent. To assess genetic heterogeneity of the TTV genome in more detail, a sequence analysis of PCR fragments (271 bp) amplified from open reading frame 1 (ORF1) was performed. PCR fragments were amplified from 5 to 40% of serum specimens obtained from patients with different forms of hepatitis who reside in different countries (e.g., China, Egypt, Vietnam, and the United States) and from normal human specimens obtained from U.S. residents. A total of 170 PCR fragments were sequenced and compared to sequences derived from the corresponding TTV genome region deposited in GenBank. Genotypes 2 and 3 were found to be significantly more genetically related than any other TTV genotype. Moreover, three sequences were shown to be almost equally related to both genotypes 2 and 3. These observations suggest a merger of genotypes 2 and 3 into one genotype, 2/3. Additionally, five new groups of TTV sequences were identified. One group represents a new genotype, whereas the other four groups were shown to be more evolutionary distant from all known TTV sequences. The evolutionary distances between these four groups were also shown to be greater than between TTV genotypes. The phylogenetic analysis suggested that these four new genetic groups represent closely related yet different viral species. Thus, TTV exists as a "swarm" of at least five closely related but different viruses. These observations suggest a high degree of genetic complexity within the TTV population. The finding of the additional TTV-related species should be taken into consideration when the association between TTV infections and human diseases of unknown etiology is studied.     

BoLA class I charge heterogeneity reflects the expression of more than two loci

Internationally recognized allo-antisera in lymphocyte microcytotoxicity assays are thought to detect allelic products of a single highly polymorphic class I locus. A recent report suggested that two bovine lymphocyte antigen (BoLA) class I loci are expressed at the protein level. However, 1D-IEF analysis of BoLA class I molecules reveals multi-band patterns which cannot be reconciled with the reported number of loci. The aim of this study was to investigate the origins of the charge diversity of BoLA class I molecules observed using 1D-IEF. BoLA class I molecules appear to be glycosy-lated at a single N-linked position with a complex type carbohydrate moiety which has up to three terminal sialic acid residues. Class I molecules immunoprecipitated from resting bovine PBL are not phosphorylated. Neither modification is responsible for the observed charge heterogeneity. Peptide mapping reveals that different BoLA charge variants have distinct digestion patterns. Furthermore, a number of different polypeptides are associated with each serological specificity. These polypeptides appear to be encoded by different loci which exist in linkage disequilibrium. The number of charge variants with different peptide maps indicates that the BoLA system has a minimum of three class I loci expressed at the protein level.     

Wood anatomy reflects the distribution of Krascheninnikovia ceratoides (Chenopodiaceae)

Wood anatomy characters of the Eurasian/North American half-shrub Krascheninnikovia ceratoides were investigated on plants from different parts of the wide distribution area. The secondary xylem is developed as result of anomalous secondary thickening. The vessels are small (commonly <50 μm) and the secondary xylem is rayless. Differences in vessel diameter exist between the main axis, basal branches and flowering shoots. The maximum as well as the minimum vessel diameter in the flowering shoots is significantly smaller compared to the basal branches, as well as in the main axis in all provenances. The length of thick-walled libriform fibre cells varies between the main axis and the basal branches of the same plant individual, but in the short main axis the length of libriform fibre cells is relatively constant (193-217 μm), independent of provenance and climate. Obviously, the length of libriform fibre cells is a conservative character and dependent on plant size. The vessel diameter is a more sensitive parameter for ecological studies. It varies more clearly, considering the vessel position in the plant axis system, in relation with climate differences of the provenances of the examined plants.Plants from a temperate, semi-humid climate have the largest vessel diameter in basal branches and flowering shoots, while the vessel diameter of plants from an arid temperate climate in Central Asia is smallest. The vessel diameters depend not only on climate, but also on the position in the axial system of the plants. There is a trade-off between average vessel number and their maximum diameter in flowering shoots of populations from Russia and Mongolia. Thus, vessel diameters depend on both, position in the axial system of the plant and climatic conditions.     

Protooncogenes subserve memory formation in the adult CNS

Studies of the signal transduction mechanisms underlying learning and memory have provided many new insights into the molecular mechanisms underlying associative conditioning in mammals. In this issue of Neuron, Gean and colleagues report the discovery that the PI-3 kinase/AKT(PKB) pathway contributes to LTP and the consolidation of amygdala-dependent cued fear conditioning in rats.     

Regulatory anatomy of the murine interleukin-2 gene.

We have cloned the mouse IL2 gene and sequenced 2800 bp of 5' flanking DNA. Comparison to the previously reported human sequence revealed extensive identity (approximately 86%) between the two genes from +1 to -580 with additional small islands of homology further upstream. Proximal sites which have been shown to be important in regulation of the human IL2 gene are well conserved in sequence and location. Transfection experiments using hybrid gene constructs containing varying lengths of the mouse 5' flanking DNA linked to a CAT reporter gene have demonstrated the presence of several novel positive and negative regulatory elements. One negative regulatory region lying between -750 and -1000 consists primarily of alternating purines and pyrimidines and is absent from the human gene. The conserved region from -321 and -578, an upstream segment from -1219 to -1332, and another region of approximately 450 bp from -1449 to -1890, which contained a well-conserved sequence of 60 bp, were each associated with enhanced levels of expression. We found no evidence for intragenic or downstream enhancer elements in this gene. All the elements identified affect only the magnitude of the inducible response, for no region when deleted had the effect of altering either the need for induction, the kinetics of stimulation, or the cell-type specificity of expression. Deletion studies suggest a strong requirement for NFAT binding even in the presence of extensive 5' flanking sequence. Therefore we conclude that IL2 gene expression is controlled primarily through a central TH1-specific signaling pathway, which acts through proximal elements, while distal cis-elements exert a secondary modulating effect.     

Defining the NG2-expressing cell of the adult CNS

The NG2 proteoglycan is believed to be an in vivomarker for oligodendrocyte progenitors found in the developing brain. The prevalence of NG2-expressing cells that remain in the adult CNS following the end of gliogenesis is significant. Current research is focused on how this cell participates in the normal function of the adult CNS and whether it may be activated by injury and/or contribute to repair. Despite substantial evidence for a sub-population of NG2-expressing cells playing a glial progenitor role in the adult CNS, there is much to be learned. Specifically, the heterogeneity of this population has not been adequately addressed for the adult CNS and while NG2 cells continue to divide in the adult CNS it is not clear what function they serve once myelination is complete. Future studies should elucidate the functional importance of NG2 in a variety of cell functions and shed light on the role NG2-expressing cells play in the intact and diseasedCNS.     

Culture of murine mastocytes: heterogeneity of the ultrastructural aspects

In cultures of normal mouse hematopoietic cells containing mast cell growth factor develop cells with many features of mast cells. These cells seem heterogeneous with respect to size, cell surface, granules maturity and morphology of nucleus using transmission and scanning electron microscopy. Alcian blue-safranin staining shows that most of the proteoglycan synthesized by cultured mast cells is weakly sulfated and non heparin-mucopolysaccharides. These results support the view that cultured mast cells resemble to mucosal mast cells, and are clearly different from serosal mast cells.     

Influence of the environment on adult CNS plasticity and repair

During developmental critical periods, external stimuli are crucial for information processing, acquisition of new functions or functional recovery after CNS damage. These phenomena depend on the capability of neurons to modify their functional properties and/or their connections, generally defined as "plasticity". Although plasticity decreases after the closure of critical periods, the adult CNS retains significant capabilities for structural remodelling and functional adaptation. At the molecular level, structural modifications of neural circuits depend on the balance between intrinsic growth properties of the involved neurons and growth-regulatory cues of the extracellular milieu. Interestingly, experience acts on this balance, so as to create permissive conditions for neuritic remodelling. Here, we present an overview of recent findings concerning the effects of experience on cellular and molecular processes responsible for producing structural plasticity of neural networks or functional recovery after an insult to the adult CNS (e.g. traumatic injury, ischemia or neurodegenerative disease). Understanding experience-dependent mechanisms is crucial for the development of tailored rehabilitative strategies, which can be exploited alone or in combination with specific therapeutic interventions to improve neural repair after damage.     

Stability and variability of synapses in the adult molluskan CNS

Synaptic transmission was examined between identified neurons in the central nervous system (CNS) of the freshwater mollusk, Lymnaea stagnalis. Four identified neurons were used: Right Pedal Dorsal one (RPeD1; a dopaminergic respiratory interneuron), Visceral Dorsal two and three (VD2/3), and Visceral Dorsal four (VD4; a cardiorespiratory interneuron). Neuron RPeD1 synapses onto both VD2/3 and VD4, while VD4 makes a reciprocal synapse onto RPeD1. When compared from animal to animal, the connections were variable in sign. Previously, we demonstrated that, in a given animal, the RPeD1 --> VD4 synapse could be either inhibitory, biphasic, or undetectable. The present study now expands this concept of variability by showing that the RPeD1 --> VD2/3 synapse was either excitatory or undetectable from animal to animal, while the synapse from VD4 to RPeD1 was observed as inhibitory, biphasic, depolarizing, excitatory, or undetectable. Next, we used 1-day organ culture to determine if the variability observed between animals is a product of ongoing change to the sign of these identified synapses and whether or not the extent of change could be influenced by the culture conditions. Changes to the sign of transmission occurred within minutes and, more commonly, after 24-h organ culture. All three synapses were investigated before and after 1-day organ culture, in either defined medium (DM) or brain-conditioned medium (CM). Regardless of culture conditions, the RPeD1 --> VD2/3 synapse showed no change of sign, i.e., it was relatively stable. However, the synapses between RPeD1 and VD4 did change sign, and when cultured in CM, the VD4 --> RPeD1 synapse changed significantly more than in DM. These data indicate that variability of some synapses reflects changes at these synapses. This is the first report that specific synapses in an adult CNS can change sign, and that the sign of transmission can be modulated by environmental conditions.     

Maintaining the neuronal phenotype after injury in the adult CNS

Multiple genetic and epigenetic events determine neuronal phenotype during nervous system development. After the mature mammalian neuronal phenotype has been determined it is usually static for the remainder of life, unless an injury or degenerative event occurs. Injured neurons may suffer one of three potential fates: death, persistent atrophy, or recovery. The ability of an injured adult neuron to recover from injury in adulthood may be determined by events that also influence neuronal phenotype during development, including expression of growth-related genes and responsiveness to survival and growth signals in the environment. The latter signals include neurotrophic factors and substrate molecules that promote neurite growth. Several adult CNS regions exhibit neurotrophic-factor responsiveness, including the basal forebrain, entorhinal cortex, hippocampus, thalamus, brainstem, and spinal cord. The specificity of neurotrophic-factor responsiveness in these regions parallels patterns observed during development. In addition, neurons of several CNS regions extend neurites after injury when presented with growth-promoting substrates. Whenboth neurotrophic factors and growth-promoting substrates are provided to adult rats that have undergone bilateral fimbria-fornix lesions, then partial morphological and behavioral recovery can be induced. Gene therapy is one useful tool for providing these substances. Thus, the mature CNS remains robustly responsive to signals that shape nervous system development, and is highly plastic when stimulated by appropriate cues.     

Intra-individual heterogeneity of rDNA allows the distinction between two closely related species in the Genus Helianthus

The Ribosomal DNAs of Helianthus annuus and H. argophyllus were analysed. Total DNA from single individuals of six cultivated lines, one wild ecotype ofH. annuus, and three ecotypes of H. argophyllus, were digested with various restriction enzymes. Hybridisation of Southern blots with sunflower ribosomal probes containing most of the interspacer regions (R3) or the 25 s coding region (R2) reveals different patterns from those expected: while no difference between H. annuus and H. argophyllus had been observed in previous rDNA RFLP analysis, our study clearly distinguished the two species on the basis of two different patterns when using R3 and BamHI, BstYI, or EcoRI/BamHI. Furthermore, the sum of the fragment weights of the BamHI restriction patterns was much greater than that of the rDNA entire unit-weight space. The co-existence of different rDNA units within single individuals is proposed as a model to explain these results. Four rDNA units were distinguished, which differed in their state of methylation and by the presence of mutations at two BamRI restriction sites. H. annuus individuals displayed two types of rDNA units while H. argophyllus individuals displayed four types.     

Effect of bench-scale culture conditions on murine IgG heterogeneity

A stable murine hybridoma cell line, secreting IgG1 antibodies (7H3) against the soluble type I receptor for Tumor Necrosis Factor (sTNF-R1), was cultivated in two different bioreactor systems, a hollow fiber and a stirred tank fermentor, in order to evaluate the effect of culture conditions on antibody structural and functional heterogeneity. Conventional serum-supplemented and serum-free media were chosen for fermentation in stirred tank bioreactor, whereas only serum-supplemented media were used for hollow fiber cultivation. Extent of glycosylation, determined by lectin binding assays, and charge heterogeneity of murine monoclonal antibodies displayed relevant variations according to the fermentation system used. After complete sugars removal by N-glycosidase F treatment, charge heterogeneity were still observed suggesting the occurrence of additional modifications at the protein level. In vitro culture in serum-supplemented media carried out with the hollow fibre system led to higher productivity but greater antibody charge heterogeneity and differences in lectin-binding profile than cultivation in the stirred tank bioreactor.Results cumulatively indicated that hybridoma cultivation methods, but also cultivation time, influence antibody heterogeneity, both in the protein and sugar moieties. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 17-25, 1997.