Surface ultrastructure of pit organ, spectacle, and non pit organ epidermis of infrared imaging boid snakes: A scanning probe and scanning electron microscopy study.

Boid snakes possess unique infrared imaging pit organs. The ultrastructure of the surfaces of these organs scatter or reflect electromagnetic radiation of specific wavelengths. Pit organ epidermal surfaces of boid snakes are covered with arrays of pore-like structures called micropits. In order to determine the dimensions of this complicated surface structure, we have performed the first ultrastructural analysis on snake epidermis by high-resolution microscopy techniques. Using scanning probe microscopy and scanning electron microscopy, we found that the epidermis of pit organ, maxillary non pit organ, spectacle, and ventral scales contain arrays of micropits. These scale surfaces also contain major surface features of overlapping plate-like structures. Pit organ micropits averaged 319 nm in diameter and 46 nm in depth and were spaced an average of 808 nm from each other. These micropits were significantly deeper, of greater diameter, and spaced at greater distances apart than those of the other scales. Plate structures of the pit organs had a mean distance between plates of 3.5 microm and a mean plate step height of 151 nm. These differences serve to strengthen the argument that arrays of micropit and plate surface structures function as spectral filters or anti-reflective coatings with respect to incident electromagnetic radiation.     

The infundibular organ of the lancelet (Branchiostoma lanceolatum,Acrania): an immunocytochemical study

Reissner's fibers are secretions produced by different ependymal areas of the chordate brain, viz., in adult vertebrates, by the dorsal subcommissural organ, and in all stages of cephalochordates (Branchiostoma lancelets), by the ventral infundibular organ. Fibers produced by these different organs are seemingly identical and the two fiber sources also share some immunocytochemical and lectin-binding properties. The secretions in these two glands are, however, not identical; the infundibular organ cells are strongly reactive with antibodies against vertebrate Reissner's fibers, but they do not react with antibodies raised against the source of the vertebrate fibers, viz., the subcommissural organ. The results support the possibility that, in adult vertebrates, the Reissner's fibers are composed of material not only from the subcommissural organ, but also from another, not yet identified, source that is identical or equivalent to the infundibular organ of the lancelet. There are indications that the infundibular organ is immunocytochemically closely akin to some secretory cells in the vertebrate embryonic brain and also to those that produce the juvenile vertebrate Reissner's fibers, viz., secretory cells in the flexural organ.     

The pineal organ as a folded retina: immunocytochemical localization of opsins

The most simple pineal complex (the pineal and parapineal organs of lampreys), consists of saccular evaginations of the diencephalic roof, and has a retina-like structure containing photoreceptor cells and secondary neurons. In more differentiated vertebrates, the successive folding of the pineal wall multiplies the cells and reduces the lumen of the organ, but the pattern of the histological organization remains similar to that of lampreys; therefore, we consider the histological structure of the pineal organ of higher vertebrates as a 'folded retina'. The cell membrane of several pineal photoreceptor outer-segments of vertebrates immunoreact with anti-retinal opsin antibodies supporting the view of retina-like organization of the pineal. Some other pineal outer segments do not react with retinal anti-opsin antibodies, a result suggesting the presence of special pineal photopigments in different types of pinealocytes that obviously developed during evolution. The chicken pinopsin, detected in the last years, may represent one of these unknown photopigments. Using antibodies against chicken pinopsin, we compared the immunoreactivity of different photoreceptors of the pineal organs from cyclostomes to birds at the light and electron microscopic levels. We found pinopsin immunoreaction on all pinealocytes of birds and on the rhodopsin-negative large reptilian pinealocytes. As the pinopsin has an absorption maximum at 470 nm, these avian and reptilian immunoreactive pinealocytes can be regarded as green-blue light-sensitive photoreceptors. Only a weak immunoreaction was observed on the frog and fish pinealocytes and no reaction was seen in cyclostomes and in the frontal organ of reptiles. Some photoreceptors of the retina of various species also reacted the pinopsin antibodies, therefore, pinopsin must have certain sequential similarity to individual retinal opsins of some vertebrates.     

Protein analysis by isoelectric focusing in a capillary array with an absorption imaging detector

Isoelectric focusing (IEF) was successuflly performed in capillary arrays with up to four capillaries. Separated proteins in the capillary array were detected by an UV absorption imaging detector. The whole analysis time for all samples in the capillary array was only 3 min due to the real-time imaging detector. The instrument was applied to analyse several protein samples including different human hemoglobin variants, myoglobin, transferrin, carbonic anhydrase and a monoclonal antibody to fluorescein. Because of good reproducibility of the focused pattern, unknown samples can be run simultaneously with a standard in the multichannel instrument and the components of unknown samples can be identified by comparing their zone positions to those of the standard. Minor components can be determined by the instrument in the presence of major components with 100 times higher concentrations in human hemoglobin samples. This instrument could be a powerful analytical tool for clinical analysis and for quality control in pharmaceutical companies.     

Nmrglue: an open source Python package for the analysis of multidimensional NMR data

Nmrglue, an open source Python package for working with multidimensional NMR data, is described. When used in combination with other Python scientific libraries, nmrglue provides a highly flexible and robust environment for spectral processing, analysis and visualization and includes a number of common utilities such as linear prediction, peak picking and lineshape fitting. The package also enables existing NMR software programs to be readily tied together, currently facilitating the reading, writing and conversion of data stored in Bruker, Agilent/Varian, NMRPipe, Sparky, SIMPSON, and Rowland NMR Toolkit file formats. In addition to standard applications, the versatility offered by nmrglue makes the package particularly suitable for tasks that include manipulating raw spectrometer data files, automated quantitative analysis of multidimensional NMR spectra with irregular lineshapes such as those frequently encountered in the context of biomacromolecular solid-state NMR, and rapid implementation and development of unconventional data processing methods such as covariance NMR and other non-Fourier approaches. Detailed documentation, install files and source code for nmrglue are freely available at http://nmrglue.com. The source code can be redistributed and modified under the New BSD license.     

Differential distribution of G-protein beta-subunits in brain: an immunocytochemical analysis.

Heterotrimeric G proteins play central roles in signal transduction of neurons and other cells. The variety of their alpha-, beta-, and gamma-subunits allows numerous combinations thereby confering specificity to receptor-G-protein-effector interactions. Using antisera against individual G-protein beta-subunits we here present a regional and subcellular distribution of Gbeta1, Gbeta2, and Gbeta5 in rat brain. Immunocytochemical specificity of the subtype-specific antisera is revealed in Sf9 cells infected with various G-protein beta-subunits. Since Gbeta-subunits together with a G-protein gamma-subunit affect signal cascades we include a distribution of the neuron-specific Ggamma2- and Ggamma3-subunits in selected brain areas. Gbeta1, Gbeta2, and Gbeta5 are preferentially distributed in the neuropil of hippocampus, cerebellum and spinal cord. Gbeta2 is highly concentrated in the mossy fibres of dentate gyrus neurons ending in the stratum lucidum of hippocampal CA3-area. High amounts of Gbeta2 also occur in interneurons innervating spinal cord alpha-motoneurons. Gbeta5 is differentially distributed in all brain areas studied. It is found in the pyramidal cells of hippocampal CA1-CA3 as well as in the granule cell layer of dentate gyrus and in some interneurons. In the spinal cord Gbeta5 in contrast to Gbeta2 concentrates around alpha-motoneurons. In cultivated mouse hippocampal and hypothalamic neurons Gbeta2 and Gbeta5 are found in different subcellular compartments. Whereas Gbeta5 is restricted to the perikarya, Gbeta2 is also found in processes and synaptic contacts where it partially colocalizes with the synaptic vesicle protein synaptobrevin. An antiserum recognizing Ggamma2 and Ggamma3 reveals that these subunits are less expressed in hippocampus and cerebellum. Presumably this antiserum specifically recognizes Ggamma2 and Ggamma3 in combinations with certain G alphas and/or Gbetas. The widespread but regionally and cellularly rather different distribution of Gbeta- and Ggamma2/3-subunits suggests that region-specific combinations of G-protein subunits mediate signal transduction in the central nervous system. The different subcellular distribution of Gbeta-subunits in cultivated neurons reflects that observed in tissue where Gbeta5 and Gbeta2 associate preferentially with the perikarya and the neuropil, respectively, and suggests an additional association of Gbeta2 with secretory vesicles.     

Association between nuclear matrix and terminal transferase: an electron microscope immunocytochemical analysis

Summary Nuclear matrix extracted from KM-3, a human pre-B leukemia cell line, appears to have a site of linkage for terminal deoxynucleotidyl transferase (TdT). The immunocytochemical analysis of the distribution of TdT using a rabbit polyclonal antibody which recognizes human terminal transferase, shows that the nuclear framework of these cells contains sites of immunoreactivity that appear uniformly distributed on the matrix fibres, while the nucleolar region is unreactive. This evidence points out the possibility that TdT could reside in the proteinaceous scaffold of the nucleus defined as nuclear matrix, thus strengthening the evidence for the metabolic and regulatory roles ascribed to this nuclear framework.     

The cell cycle of glial cells grown in vitro: an immunocytochemical method of analysis.

Studies of cell cycles have traditionally employed [3H]- and [14C]-thymidine to label the DNA of proliferating cells and autoradiography to reveal the thymidine label. The development of antibodies to the thymidine analogue 5-bromodeoxyuridine (BrdU) has allowed the development of an immunocytochemical method analogous to the thymidine autoradiographic technique. In direct comparisons, we found that the immunocytochemical method consistently detected a larger number of proliferating cells. This suggests that it may be a more sensitive index of proliferation than thymidine autoradiography in some systems. We used the BrdU method to analyze the cycle of astroglia cultured from neonatal mouse cerebral cortex. Cells were exposed to BrdU for 1 hr to label a discrete subpopulation of proliferating cells. At 2-36 hr after the pulse, a combination of anti-BrdU immunocytochemistry and counterstaining with propidium iodide was used to identify proliferating cells. The length of the cell cycle was determined by charting the percent of BrdU-labeled mitotic cells vs time after the pulse. We found the average length of the cell cycle of astrocytes grown in vitro to be 20.5 hr. The combined G2 + M phases were 2-3 hr. These values are virtually identical with those found for glial cells in vivo, suggesting that the culture environment does not interfere with the normal control of cell cycle length.     

GABAergic system of the pineal organ of an elasmobranch (Scyliorhinus canicula): a developmental immunocytochemical study

The present immunocytochemical study provides evidence of a previously unrecognized, rich, γ-aminobutyric acid (GABA)-ergic innervation of the pineal organ in the dogfish (Scyliorhinus canicula). In this elasmobranch, the pineal primordium is initially detected at embryonic stage 24 and grows to form a long pineal tube by stage 28. Glutamic acid decarboxylase (GAD)-immunoreactive (-ir) fibers were first observed at stage 26, and by stage 28, thin GAD-ir fibers were detectable at the base of the pineal neuroepithelium. In pre-hatchling embryos, most fibers gave rise to GAD-ir boutons that were localized in the basal region of the neuroepithelium, although a smaller number of labeled terminals ascended to the pineal lumen. A few pale GAD-ir perikarya were observed within the pineal organ of stage 29 embryos, but GAD-ir perikarya were not observed at other developing stages or in adults. In contrast, GABA immunocytochemistry revealed the presence of GABAergic perikarya and fibers in the pineal organ of late stage embryos and adults. Although high densities of GABAergic cells were observed in the paracommissural pretectum, posterior tubercle, and tegmentum of dogfish embryos (regions previously demonstrated to contain pinealopetal cells), the presence of GABA-ir perikarya in the pineal organ strongly suggests that the rich GABAergic innervation of the elasmobranch pineal organ is intrinsic. This contrasts with the central origin of GABAergic fibers in the pineal gland of some mammals. This work was supported by the Spanish Education and Science Ministry and FEDER (BXX2000-0453-C02 and BFU2004-03313/BF1), the Xunta de Galicia (PGIDT99BIO20002), and NIH/NIDCD awards R01 DC01705 and P01 DC01837 (to G.R.H.).     

Association between nuclear matrix and terminal transferase: an electron microscope immunocytochemical analysis

Nuclear matrix extracted from KM-3, a human pre-B leukemia cell line, appears to have a site of linkage for terminal deoxynucleotidyl transferase (TdT). The immunocytochemical analysis of the distribution of TdT using a rabbit polyclonal antibody which recognizes human terminal transferase, shows that the nuclear framework of these cells contains sites of immunoreactivity that appear uniformly distributed on the matrix fibres, while the nucleolar region is unreactive. This evidence points out the possibility that TdT could reside in the proteinaceous scaffold of the nucleus defined as nuclear matrix, thus strengthening the evidence for the metabolic and regulatory roles ascribed to this nuclear framework.     

The endocrine pancreas of Triturus cristatus: an immunocytochemical study

The endocrine pancreas of Triturus cristatus carnifex was studied with the aid of immunocytochemical methods, showing cells immunoreactive to anti-insulin serum (B cells), a small population of cells immunoreactive to anti-glucagon serum only (A cells), rare cells positive to anti-PP serum only (PP or F cells), and a larger population of cells immunoreactive both to anti-glucagon and to anti-PP sera. B cells lied in the core of the islet, while the A/PP cells were located at the periphery, forming digitations extending into the exocrine parenchyma. D cells were present in small number in the islet while they were more numerous scattered in the exocrine parenchyma. A/PP cells as well as D cells showed one or two long cytoplasmic extensions often in contact with blood vessels.     

Genetic analysis of an osmotic sensitive

Summary The genetic analysis of VY1160 sorbitol dependent, osmotic sensitive yeast mutant led to the identification of three different nuclear recessive mutations. Two of them, designated sorb^- and ts1 are closely linked to one another. The mutation sorb^- determines the lysis, while the mutation tsl increases the ability for lysis of the sorbitol dependent cells. The third mutation ts2 segregates independently from the other two and confers the sensitivity of VY1160 mutant cells towards rifampicin.     

Sarment: Python modules for HMM analysis and partitioning of sequences

Sarment is a package of Python modules for easy building and manipulation of sequence segmentations. It provides efficient implementation of usual algorithms for hidden Markov Model computation, as well as for maximal predictive partitioning. Owing to its very large variety of criteria for computing segmentations, Sarment can handle many kinds of models. Because of object-oriented programming, the results of the segmentation are very easy tomanipulate.     

Community genomic analysis of an extremely acidophilic sulfur-oxidizing biofilm

Highly acidic (pH 0–1) biofilms, known as ‘snottites'', form on the walls and ceilings of hydrogen sulfide-rich caves. We investigated the population structure, physiology and biogeochemistry of these biofilms using metagenomics, rRNA methods and lipid geochemistry. Snottites from the Frasassi cave system (Italy) are dominated (>70% of cells) by Acidithiobacillus thiooxidans, with smaller populations including an archaeon in the uncultivated ‘G-plasma'' clade of Thermoplasmatales (>15%) and a bacterium in the Acidimicrobiaceae family (>5%). Based on metagenomic evidence, the Acidithiobacillus population is autotrophic (ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), carboxysomes) and oxidizes sulfur by the sulfide–quinone reductase and sox pathways. No reads matching nitrogen fixation genes were detected in the metagenome, whereas multiple matches to nitrogen assimilation functions are present, consistent with geochemical evidence, that fixed nitrogen is available in the snottite environment to support autotrophic growth. Evidence for adaptations to extreme acidity include Acidithiobacillus sequences for cation transporters and hopanoid synthesis, and direct measurements of hopanoid membrane lipids. Based on combined metagenomic, molecular and geochemical evidence, we suggest that Acidithiobacillus is the snottite architect and main primary producer, and that snottite morphology and distributions in the cave environment are directly related to the supply of C, N and energy substrates from the cave atmosphere.     

Population distribution of aerobic extremely thermophilic microorganisms in an icelandic natural hot spring

Summary The aerobic microbial population of natural hot spring water was investigated by means of taxonomic analyses of 33 characteristic properties. Twenty-eight strains isolated in Iceland were compared with 3 Thermus and 4 Bacillus reference strains. The results showed a high variability in the characteristics of bacteria isolated from nearby sampling areas. No typical sampling-area-specific or temperature-specific populations were found. These findings suggested high diversity of thermophiles due to environmental factors. In this report adaptation mechanisms are postulated and discussed in relation to the growth behaviour of Thermus strains in the natural environment and in continuous cultivation.     

The ovotestis: an underdeveloped organ of evolution

In animals that have separate sexes (gonochorists), many sperm are produced to fertilise a few eggs. As the male germline undergoes more mitoses, so the accumulated mutation frequency is elevated in sperm compared with ova, and evolution is 'male-driven'. In contrast, in many hermaphroditic animals, a single organ--the ovotestis--produces both ova and sperm. Since self-renewing cells in the ovotestis may give rise to both cell types throughout life, ova in hermaphrodites could in theory have undergone as many cell divisions as sperm. Here, I consider some possible effects of the ovotestis on evolution. In particular, I hypothesise that the accumulated mutation frequency of nuclear genes in hermaphrodites (including species that change sex) may reach twice that compared with gonochorists. There may be an even greater increase in the mitochondrial mutation frequency. Further developmental studies and the accumulation of comparative data should allow hypothesis testing. If the prediction is correct, then it may provide the most-straightforward explanation for the extraordinary diversity of mitochondrial DNA in some hermaphrodites, especially molluscs.     

The nervous system of the chicken proventriculus: an immunocytochemical and ultrastructural study

The proventriculus constitutes the glandular region of the chicken stomach. This organ is innervated by two parasympathetic networks, the myenteric and submucous plexus, and here we present a systematic study of this system by immunohistochemistry and electron microscopy. All the neurons and fibres were positive for the neural markers, protein gene product 9.5 and the amidating enzymes. Immunoreactivities for the constitutive neuronal isoform of the enzyme nitric oxide synthase and the vasoactive intestinal peptide were present in neuronal bodies suggesting an intrinsic origin for the similarly immunoreactive fibres found in the proventriculus. On the other hand, immunoreactivity to gastric inhibitory peptide was only found in varicose fibres making contact with the blood vessels and the glandular epithelium, but never in the neuronal somas, suggesting that this substance may be provided by an extrinsic nervous system whose neuronal bodies are located elsewhere. Electron microscopy revealed frequent neuromuscular and neuroepithelial connections in the muscle layers, the wall of the blood vessels and the epithelium. In addition, synapsis-like structures were identified in the proximity of cells belonging to the diffuse endocrine system, providing a new example of neuroendocrine contacts. No positivity was found for antibodies against other neural substances including somatostatin, peptide histidine–isoleucine, peptide tyrosine–tyrosine, neuropeptide tyrosine, bombesin, met-enkephalin, serotonin, substance P, galanin, calcitonin gene-related peptide and S-100 protein.     

The reaction cycle of bacteriorhodopsin: an analysis using visible absorption,photocurrent and infrared techniques

The light activated absorbance changes and photo-electric events of bacteriorhodopsin (bR) were simultaneously measured. The results were compared with the kinetics of the time resolved infrared signals which are characteristic for protonation changes of Asp residues, chromophore vibrations, and amide I vibrations. Each data set was analyzed separately. Assuming first order reactions the experimental curves in the time range from L back to bR could be fitted by a sum of five exponentials. However, for the photocurrent signal only four exponentials were necessary. The corresponding half-life times were of the same order of magnitude. Simultaneous fits of the traces from absorption changes in the visible range and the photocurrent signal provided evidence that the photocurrent data could also be described by the same sum of exponentials as the data obtained in the visible range. The rate constants obtained from the different methods applied were, within the limits of error, identical. These results demonstrate that retinal monitors not only charge displacements but also conformational movements of the protein moiety. The reprotonation of the Schiff base occurs synchronously with a protonation change of an internal aspartic acid which absorbs at 1755 cm^–1. From the IR-signals, amplitude spectra could be derived which provided evidence that Asp-residues absorbing at 1765 cm^–1 (Asp85) and 1755 cm^–1 are still protonated in the O-intermediate. Major conformational changes of the peptide back bone occur in the time range of the L M transition and with opposite sign during the decay of the O-intermediate. Offprint requests to: M. Engelhard     

The efficiency of mispaired ligations by lambda integrase is extremely sensitive to context

The integrase protein (Int) of phage lambda is a well-studied representative of the tyrosine recombinase family, whose defining features are two sequential pairs of DNA cleavage/ligation reactions that proceed via a 3' phosphotyrosine covalent intermediate to first form and then resolve a Holliday junction recombination intermediate. We devised an assay that takes advantage of DNA hairpin formation at one Int target site to trap Int cleavages at a different target site, and thereby reveal iterative cycles of cleavage and ligation that would otherwise be undetected. Using this assay and others to compare wild-type Int and a mutant (R169D) defective in forming proper dimer/tetramer interfaces, we found that the efficiency of "bottom-strand" DNA cleavage by wild-type Int, but not R169D, is very sensitive to the base-pair at the "top-strand" cleavage site, seven base-pairs away. We show that this is related to the finding that hairpin formation involving ligation of a mispaired base is much faster for R169D than for wild-type Int, but only in the context of a multimeric complex. During resolution of Holliday junction recombination intermediates, wild-type Int, but not R169D, is very sensitive to homology at the sites of ligation. A long-sought insight from these results is that during Holliday junction resolution the tetrameric Int complex remains intact until after ligation of the product helices has been completed. This contrasts with models in which the second pair of DNA cleavages is a trigger for dissolution of the recombination complex.