Immunoreactivity for calcium-binding proteins in the claustrum of the monkey

The sequence of degenerative changes in the retinal pigment epithelium (RPE) and the choroid of retinal degeneration (rd)-mice was studied in correlation with photoreceptor changes. Three weeks to 26-month-old animals were investigated using light and transmission electron microscopy, enzyme histochemistry and quantitative morphology. Changes in the choriocapillaris (CC) were additionally studied by scanning electron microscopy of corrosion cast preparations. In 3-week-old mice, in which most of the outer segments of photoreceptors in the central portion of the retina had disappeared but remnants of the cells were still present, the RPE was enlarged and showed elongated microvilli. In 8-week-old animals, the photoreceptors were completely absent in large areas of the posterior pole region. In these areas the RPE was also completely lost. Quantitative evaluation performed in histological serial sections showed that loss of RPE measured as length of RPE-free Bruch's membrane, continuously increased up to the age of 20 months. In 8-week-old animals, CC adjacent to degenerating RPE showed loss of fenestration. In 10-week-old animals, the CC disappeared in those areas where the RPE was already lacking. The loss of CC increased with increasing age and in 20-month-old animals 5-10% of the entire CC was lacking. Loss of the related arterioles and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d)-positive nerve fibers occurred only in approximately 2-year-old rd-mice. Compared to other animal models, RPE and CC defects in rd-mice are relatively large. The rd-mice might therefore provide a good tool to study factors involved in CC degeneration.     

Morphological features of neurons containing calcium-binding proteins in the human striatum

An immunohistochemical approach was used to characterize the morphological phenotype of neurons containing the calcium-binding proteins calretinin (CR), parvalbumin (PV), or calbindin-D28k (CB) in the normal human striatum. The protein CR occurs in at least four morphologically distinct types of neurons. Apart from the numerous medium-sized aspiny interneurons and the less abundant giant aspiny interneurons, CR also labels some medium-sized spiny neurons morphologically identical to striatal projection neurons. This finding indicates that CR is not only confined to striatal interneurons but also may be involved in the function of certain projection neurons. Some small and peculiar bushy-like aspiny neurons also are enriched with CR. These neurons could correspond to the dwarf or neurogliform neurons first described by Ramón y Cajal (1911). Three types of PV-immunoreactive striatal neurons can be visualized in the human striatum: 1) the common medium-sized aspiny leptodendritic neurons, 2) some smaller and profusely arborized aspiny neurons, and 3) a few large and intensely stained neurons with conspicuously beaded and poorly branched dendrites. The protein CB labels virtually all medium-sized spiny projection neurons located in the striatal matrix but also identifies a small subset of large and more intensely immunostained aspiny neurons. The latter finding indicates that CB is not entirely confined to striatal projection neurons but also may play a role in local circuit neurons. These normative data should help our understanding of the chemical anatomy of the human striatum in both health and disease.     

Fluorescence detection of proteins and amino acids in capillary electrophoresis using a post-column sheath flow reactor

A simple laser-induced fluorescence detection method for proteins and amino acids in capillary electrophoresis is reported. A sheath flow cell is utilized as a post-column reactor for fluorescence derivatization of proteins and amino acids by addition of o-phthaldialdehyde-2-mercaptoethanol to the sheath fluid. With the use of a 50 microns I.D. capillary, the limits of detection for carbonic anhydrase are 0.73 nM or 1.8 amol which represents a five- and two-fold improvement, respectively, over the best results previously reported for post-column detection. In addition, separation efficiencies up to 8.07 x 10(5) are achieved and the detector response is linear over three-orders of magnitude. These results demonstrate that mixing is adequate and the reaction kinetics are rapid enough to provide sensitive detection with this approach. Also, because this post-column derivatization scheme requires no instrumental changes to a typical sheath flow cell detector, the system can be used for detection of pre-column labeled analytes and for native fluorescence detection.     

Calretinin in the cat retina: colocalizations with other calcium-binding proteins, GABA and glycine

Regeneration of skeletal muscle was studied in the sea bream Sparus aurata, in which extensive post-larval muscle hyperplasia contributes to its large adult size, and in the zebrafish Brachydanio rerio, which shows little post-larval hyperplasia and reaches only a small adult size. Small mechanical lesions of body wall muscle were made under general anaesthesia, and the progress of subsequent regeneration was assessed at various intervals by histology and electron microscopy (for general morphology), by immunostaining for desmin and myosin isoforms (to identify the phenotype of new fibres), and by 5'-bromo-2'-deoxyuridine (BrdU) incorporation (to identify proliferating cells). Despite the difference in normal growth-related hyperplasia in these fish, a vigorous regeneration occurred in both species, giving rise to new fibres with an initial myosin composition that differed from that in mature fast-white fibres. However, species differences in myosin expression in these fibres suggest that they may have derived from different myoblast populations. In sea bream, myosin expression in regenerating fibres resembled that seen in new fibres produced in post-larval white muscle, whereas in the zebrafish it resembled that of the primitive monolayer fibres formed during embryonic development. Subsequently, most regenerating fibres gradually transformed into the mature fast-white phenotype in both species.     

Postnatal development of calcium-binding proteins calbindin and parvalbumin in human visual cortex

In adult primate visual cortex, the calcium-binding proteins calbindin (CB) and parvalbumin (PV) are localized in different subsets of GABAergic neurons with a characteristic laminar distribution. However, the emergence and development of CB and PV in relation to the periods of functional maturation of the human visual cortex are not known. Therefore, we examined (i) postnatal changes in the distribution of immunoreactivity (ir) for CB and PV in the visual cortex; (ii) the pattern of changes in immunoreactivity in relation to the synaptic maturation; and (iii) differences in the maturation of CB and PV immunoreactivity between areas 17 and 18. We found a consistently high expression of CB in neonatal visual cortex, particularly in layer IV and infragranular layers. However, despite an early appearance of PV, its peak in development occurred only after 2 months of age, characterized by a transient overexpression in the thalamo-recipient layer IV and a continuous inside-out maturation in supragranular layers. The neonatal pattern of high CB-ir in layers IV-VI was transformed during infancy and childhood into an adult pattern of high CB-ir in layer II, but low CB-ir in layer IV and infragranular layers. There was no difference in pattern and tempo of maturation of calcium-binding proteins between area 17 and 18, indicating simultaneous development of cortical inhibitory circuits among cytoarchitectonically and functionally distinct cortical areas. In addition, the reorganization of CB/PV expression temporally and spatially coincides with the course of cortical synaptogenesis, and delineates the major stages of maturation of the human visual cortex.     

Calcium regulation of Ndr protein kinase mediated by S100 calcium-binding proteins

Ndr is a nuclear serine/threonine protein kinase that belongs to a subfamily of kinases identified as being critical for the regulation of cell division and cell morphology. The regulatory mechanisms that control Ndr activity have not been characterized previously. In this paper, we present evidence that Ndr is regulated by EF-hand calcium-binding proteins of the S100 family, in response to changes in the intracellular calcium concentration. In vitro, S100B binds directly to and activates Ndr in a Ca2+-dependent manner. Moreover, Ndr is recovered from cell lysates in anti-S100B immunoprecipitates. The region of Ndr responsible for interaction with Ca2+/S100B is a basic/hydrophobic motif within the N-terminal regulatory domain of Ndr, and activation of Ndr by Ca2+/S100B is inhibited by a synthetic peptide derived from this region. In cultured cells, Ndr is rapidly activated following treatment with Ca2+ ionophore, and this activation is dependent upon the identified Ca2+/S100B-binding domain. Finally, Ndr activity is inhibited by W-7 in melanoma cells overexpressing S100B, but is unaffected by W-7 in melanoma cells that lack S100B. These results suggest that Ndr is regulated at least in part by changes in the intracellular calcium concentration, through binding of S100 proteins to its N-terminal regulatory domain.     

Hippocampal interneurons expressing glutamic acid decarboxylase and calcium-binding proteins decrease with aging in Fischer 344 rats

Aging leads to alterations in the function and plasticity of hippocampal circuitry in addition to behavioral changes. To identify critical alterations in the substrate for inhibitory circuitry as a function of aging, we evaluated the numbers of hippocampal interneurons that were positive for glutamic acid decarboxylase and those that expressed calcium-binding proteins (parvalbumin, calbindin, and calretinin) in young adult (4-5 months old) and aged (23-25 months old) male Fischer 344 rats. Both the overall interneuron population and specific subpopulations of interneurons demonstrated a commensurate decline in numbers throughout the hippocampus with aging. Interneurons positive for glutamic acid decarboxylase were significantly depleted in the stratum radiatum of CA1, the strata oriens, radiatum and pyramidale of CA3, the dentate molecular layer, and the dentate hilus. Parvalbumin interneurons showed significant reductions in the strata oriens and pyramidale of CA1, the stratum pyramidale of CA3, and the dentate hilus. The reductions in calbindin interneurons were more pronounced than other calcium-binding protein-positive interneurons and were highly significant in the strata oriens and radiatum of both CA1 and CA3 subfields and in the dentate hilus. Calretinin interneurons were decreased significantly in the strata oriens and radiatum of CA3, in the dentate granule cell and molecular layers, and in the dentate hilus. However, the relative ratio of parvalbumin-, calbindin-, and calretinin-positive interneurons compared with glutamic acid decarboxylase-positive interneurons remained constant with aging, suggesting actual loss of interneurons expressing calcium-binding proteins with age. This loss contrasts with the reported preservation of pyramidal neurons with aging in the hippocampus. Functional decreases in inhibitory drive throughout the hippocampus may occur due to this loss, particularly alterations in the processing of feed-forward information through the hippocampus. In addition, such a profound alteration in interneuron number will likely alter inhibitory control of excitability and neuronal synchrony with behavioral states.     

Coexistence of calcium-binding proteins in vagal and glossopharyngeal sensory neurons of the rat

The presence and coexistence of the calcium-binding proteins (CaBPs), calbindin D-28k, parvalbumin and S100 protein, were immunohistochemically examined in the glossopharyngeal and vagal sensory ganglia, the carotid body and taste buds. The CaBPs were found in each ganglion with the nodose ganglion containing the largest number of CaBP-immunoreactive (ir) cells (calbindin D-28k > or = S100 > parvalbumin). The coexistence of CaBPs was found in neurons of the nodose, petrosal, and jugular ganglia. Calbindin D-28k-ir neurons in the nodose and petrosal ganglia frequently colocalized S100-ir whereas calbindin D-28k-ir neurons in the jugular ganglion less frequently contained S100-ir. Only small percentages of calbindin D-28k-ir neurons in each ganglion colocalized parvalbumin. Similarly, S100-ir neurons in the nodose and petrosal ganglia frequently colocalized calbindin D-28k-ir whereas S100-ir neurons in the jugular ganglion less frequently contained calbindin D-28k-ir. Moderate to small percentages of S100-ir neurons in each ganglion colocalized parvalbumin. Parvalbumin-ir neurons nearly always colocalized S100-ir in the nodose, petrosal and jugular ganglia. Moderate to small percentages of parvalbumin-ir neurons in each ganglion colocalized calbindin D-28k. Whereas calbindin D-28k- and S100-ir were colocalized in nerve fibers and cells within taste buds of circumvallate papilla of the tongue, the coexistence of these CaBPs could not be determined in the carotid body. These findings suggest a co-operative role for CaBPs in the functions of subpopulations of nodose and petrosal ganglia neurons.     

Intracellular distributions and putative functions of calcium-binding proteins in the bullfrog vestibular otolith organs

Hair cells in the bullfrog vestibular otolith organs were immunolabeled by monoclonal and polyclonal antisera against calbindin (CaB), calmodulin (CaM), calretinin (CaR), and parvalbumin (PA). S-100, previously shown to immunolabel striolar hair cells in fish vestibular organs, only weakly immunolabeled hair cells in the bullfrog vestibular otolith organs. Immunolabeling was not detected in supporting cells. With the exception of CaR, myelinated axons and unmyelinated nerve terminals were immunolabeled by all of the above antisera. Immunolabeling was seen in all saccular hair cells, although hair cells at the macular margins were immunolabeled more intensely for CaB, CaM, and PA than more centrally located hair cells. As the macula margins are known to be a growth zone, this labeling pattern suggests that marginal hair cells up-regulate their calcium-binding proteins during hair cell development. In the utriculus, immunolabeling for CaM and PA was generally restricted to striolar hair cells. CaR immunolabeling was restricted to the stereociliary array. Immunolabeling for other calcium-binding proteins was generally seen in both the cell body and hair bundles of hair cells, although this labeling was often localized to the stereociliary array and the apical portion of the cell body. CaM and PA immunolabeling in the stereociliary array in saccular and utricular striolar cells suggests a functional role for these proteins in mechanoelectric transduction and adaptation.     

Fluorescence line narrowing applied to the study of proteins

Fluorescence line narrowing is a high resolution spectroscopic technique that uses low temperature and laser excitation to optically select specific subpopulations from the inhomogeneously broadened absorption band of the sample. When applied to the study of fluorescent groups in proteins one can obtain vibronically resolved spectra, which can be analyzed to give information on spectral line shapes, vibrational energies of both the ground and excited state molecule, and the inhomogeneous distribution function of the electronic transitions. These parameters reveal information about the chromophoric prosthetic group and the protein matrix and are functions of geometric strains and local electric fields imposed by the protein. Examples of the use of fluorescence line narrowing are discussed in investigations of heme proteins, photosynthetic systems and tryptophan-containing proteins.     

Neuronal subpopulations of developing rat hippocampus containing different calcium-binding proteins behave distinctively in trimethyltin-induced neurodegeneration

The present study investigates, by immunocytochemistry, the behavior of different neuronal subpopulations of the developing rat hippocampus, selectively labeled by the calcium-binding proteins calbindin D28-k (CB), parvalbumin (PV), and calretinin (CR), in neurodegenerative processes induced by the neurotoxicant trimethyltin (TMT). Previous studies on adult rats indicated that CB-immunoreactive (IR) neurons were affected by TMT, while PV- and CR-IR neurons were selectively spared. The present findings show that only CR-IR neurons are spared in developing rats, and in addition the number of CR-IR neurons are significantly higher in the DG of treated animals. On the contrary, PV-IR neurons, spared in adult rats, were affected by TMT during development. CB-IR neurons were affected also in developing rats, as in adults. The different postnatal time-courses of calcium-binding protein expression in relationship to the time of TMT administration (presence of CR but absence of PV) could have a role in the different behavior of CR- and PV-IR cells in developing rats.     

Cocaine adversely affects development of cortical embryonic neurons in vitro: immunocytochemical study of calcium-binding proteins

Neurons of cerebral cortex from 15-16 day old embryos of white rats (Sprague-Dawley) were cultured in MEM enriched with 5% horse serum. On the 7th day after plating the cultures were divided into three experimental and one control groups (6-8 Petri dishes in each group). In group 1, cultures were grown without additives. In group 2, cocaine chloride was added at concentrations 0.3, 0.6 and 1 mg/ml of culture. In group 3, a monoclonal antibody against calcium-binding proteins, parvalbumin (APV) or calbindin (ACB) was added at a concentration 25 microl/ml. In group 4, a combination of cocaine +APV was added at a concentration 1 mg+25 microl/ml of culture media. On the 10th day cultures were immunostained using APV and ACB antibodies. In developing GABAergic neurons of group 2 cocaine produced cytotoxic effects that were expressed in drastic decrease in number of neurons and in degeneration of their processes. The lower concentrations of cocaine caused milder cytotoxity and their effects were reversible. The highest concentration of cocaine caused irreversible degeneration of neurons. Similar cytotoxity was caused by APV or ACB in group 3. The most severe cytotoxic effects were seen in group 4, where a mixture of cocaine and APV was used. Overall, it can be concluded that cocaine in higher concentrations directly affects development of GABAergic neurons in vitro.     

Fluorescence and the structure of proteins. XXII. Fluorescence of aminotyrosyl residues formed in ribonuclease A

1. Tyrosyl residues on ribonuclease A were nitrated with tetranitromethane and then reduced to aminotyrosyl residues. By variation of reaction conditions and degree of exposure of tyrosyl residues it was possible to convert from 1 to all 6 tyrosyl to aminotyrosyl residues. 2. At the lower levels of 1-3 aminated tyrosyl residues/molecule the change in conformation seemed minor and 70% of the enzymatic activity was retained. When the three buried tyrosyl residues or all six residues were aminated only 5% of the enzymatic activity was retained. 3. Titration data, susceptibility to urea denaturation, and fluorescence characteristics indicated that some of the aminotyrosyl residues were buried in the interior and others were exposed on the surface of the protein. On the basis of the activation/emission wavelengths it was possible to distinguish buried (288/320 nm) and exposed (288/365-395 nm) aminotyrosyl residues as well as exposed tyrosyl residues (275-305 nm). 4. The modification of specific tyrosyl residues on a protein to aminotyrosyl residues appears to have some promise for observation of changes in environment of the residues that accompany various conformation changes by monitoring the fluorescence.     

Radiosensitizing and toxic properties of quinoline and nitroquinoline complexes of platinum [PtCl2(NH3)quinoline]

The assessment of drugs designed to be useful in the eradication of hypoxic (resistant) cells involves comparison of hypoxic and aerobic radiosensitization, cytotoxicities, as well as DNA binding and reduction potentials. Three pairs of isomers of quinoline complexes [amino dichloro quinoline platinum (II)] were studied in this context. For the cis 5- and 6-nitroquinoline complexes, the DNA binding and toxicity were higher with the 5-substituted ligand. Reduction potentials were similar (-260 and -280 mV). No selectivity for hypoxic toxicity was observed, but radiosensitizing ability by both complexes was greater in hypoxic (than oxic) Chinese hamster ovary (CHO) cells. The trans 5-nitroquinoline complex produced better sensitization in hypoxia than its cis isomer [enhancement ratio (ER) 1.7 at 10 microM versus 40 microM for cis]. However, this was accompanied by some aerobic sensitization. The trans isomer of the (unsubstituted) quinoline complex was considerably more toxic than its cis isomer. Neither showed selectivity for hypoxia, either as radiosensitizers or as cytotoxins, which may be attributable to the lack of a reducible (nitro) function. Four quinoline complexes showed high activity in cisplatin-resistant L1210 cells, with the lowest resistance factor being for the trans quinoline complex. Results suggest that trans complexes with one aromatic ring may have activity different from the cis geometry, which should be exploited with respect to cisplatin resistance and cross-resistance with radiation.     

P11, a unique member of the S100 family of calcium-binding proteins, interacts with and inhibits the activity of the 85-kDa cytosolic phospholipase A2

Using a two hybrid system screen of a human cDNA library, we have found that p11, a unique member of the S100 family of calcium-binding proteins, interacts with the carboxyl region of the 85-kDa cytosolic phospholipase A2 (cPLA2). p11 synthesized in a cell-free system interacts with cPLA2 in vitro. The p11-cPLA2 complex is detectable from a human bronchial epithelial cell line (BEAS 2B). Furthermore, p11 inhibits cPLA2 activity in vitro. Selective inhibition of p11 expression in the BEAS 2B cells by antisense RNA results in an increased PLA2 activity as well as an increased release of prelabeled arachidonic acid. This study demonstrates a novel mechanism for the regulation of cPLA2 by an S100 protein.     

Postnatal expression pattern of calcium-binding proteins in organotypic thalamic cultures and in the dorsal thalamus in vivo

We have investigated the influence of the chemical structure and charge of the hydrophillic headgroup on the miscibility of saturated phospholipids with acyl chain lengths differing by two methylene units, namely DMPA/DPPA, DMPC/DPPC, DMPE/DPPE and DMPG/DPPG (0.1 M NaCl). All four mixtures were analysed by DSC at pH 7. To study the influence of a change in headgroup charge, we additionally investigated DMPA/DPPA mixtures at pH 4 and 12, and DMPG/DPPG mixtures at pH 2. The experimental DSC thermograms were fitted using methods described before [Johann et al., Biophys. J. 71 (1996), 3215-3228] to obtain the temperatures of onset and end of melting and first approximations for the non-ideality parameters as a function of composition. The resulting phase diagrams were then fitted using a four non-ideality parameter model for non-ideal, non-symmetric mixing in both phases. The phase diagram of the system DMPG/DPPG has a lens-like shape, the non-ideality parameters rhog and rhol for the gel and the liquid-crystalline phase, respectively, are zero, indicating ideal mixing in both phases. For the other mixtures, differences in miscibility are observed depending on the structure of the headgroup. At pH 7, rhog > rhol, i.e., the miscibility in the liquid-crystalline phase is more ideal than in the gel state. All rhog values are positive and the sequence for rhog observed is PA>PE>PC>PG. Partial protonation of PA at pH 4 or complete deprotonation at pH 12 leads to negative non-ideality parameters for both phases, indicating a preference for mixed pair formation. Protonation of PG in DMPG/DPPG mixtures at pH 2 leads to positive non-ideality parameters for both phases, indicating a tendency for demixing. The results show, that the miscibility of phospholipids with identical headgroups but chain lengths differing by two methylene groups is dependent on headgroup structure and on headgroup charge.     

cDNA cloning of S100 calcium-binding proteins from bovine periodontal ligament and their expression in oral tissues

The periodontal ligament (PDL) is a unique tissue that is crucial for tooth function. However, little is known of the molecular mechanisms controlling PDL function. To characterize PDL cells at the molecular level, we constructed a cDNA library from bovine PDL tissue. We then focused on the isolation of S100 calcium-binding proteins (CaBPs), because they mediate Ca2+ signaling and control important cellular processes such as differentiation and metabolism. We screened the PDL cDNA library with a mouse S100A4 cDNA, and cloned the bovine cDNAs of two S100 CaBPs (S100A4 and S100A2). In northern blotting analysis, the highest expression of S100A4 was detected in PDL from erupted teeth (PDLE). PDL from teeth under eruption (PDLU) showed a lower expression of S100A4, and its expression in gingiva was faintly detectable. S100A4 expression was also high in the pulp tissue followed by the dental papilla of the tooth germ. S100A2 expression was high in PDLE and gingiva. Interestingly, only PDLE exhibited a high expression of both S100A4 and S100A2. PDLE also expressed the highest level of beta-actin, a target cytoskeletal protein for S100A4. It is conceivable that the high expression of S100A4 in PDLE is a result of the maturation of the PDL and/or a response to mechanical stress generated by mastication. Since there was a marked difference of S100A4 expression between PDL and gingiva, we propose that S100A4 could be a useful marker for distinguishing cells from these two tissues.     

Vagal and spinal afferent innervation of the rat esophagus: a combined retrograde tracing and immunocytochemical study with special emphasis on calcium-binding proteins

Vagal afferent neurons contain a variety of neurochemical markers and neuroactive substances, most of which are present also in dorsal root ganglion cells. To test for the suitability of the calcium-binding protein calretinin as a specific marker for vagal afferent fibers in the periphery, immunocytochemistry for this protein was combined with retrograde tracing. Nerve fibers in the rat esophagus, as well as vagal and spinal sensory neurons innervating the esophagus, were investigated for co-localization of calretinin with calbindin, calcitonin gene-related peptide, and NADPH diaphorase. The results indicated that calretinin immunocytochemistry demonstrates neuronal structures known as vagal afferent from other studies, in particular intraganglionic laminar endings. A few enteric neurons whose distribution was unrelated to intraganglionic laminar endings also stained for calretinin. Strikingly, calretinin immunoreactivity was absent from spinal afferent neurons innervating the rat esophagus. In intraganglionic laminar endings and nodose ganglion cells calretinin was highly co-localized with calbindin but not with calcitonin gene-related peptide. On the other hand, calbindin was also found in spinal afferents to the esophagus where it was co-localized with calcitonin gene-related peptide. Vagal afferent neurons innervating the esophagus were never positive for NADPH diaphorase. Thus, calretinin appears to be a more specific marker for vagal afferent structures in the esophagus than calbindin, which is expressed by both vagal and spinal sensory neurons. Calretinin immunocytochemistry may be utilized as a valuable tool for investigations of subpopulations of vagal afferents in certain viscera.     

Nuclear detection of cellular retinoic acid binding proteins I and II with new antibodies

Apart from the retinoic acid nuclear receptor family, there are two low molecular weight (15 kD) cellular retinoic acid binding proteins, named CRABPI and II. Mouse monoclonal and rabbit polyclonal antibodies were raised against these proteins by using as antigens either synthetic peptides corresponding to amino acid sequences unique to CRABPI or CRABPII, or purified CRABP proteins expressed in E. coli. Antibodies specific for mouse and/or human CRABPI and CRABPII were obtained and characterized by immunocytochemistry and immunoblotting. They allowed the detection not only of CRABPI but also of CRABPII in both nuclear and cytosolic extracts from transfected COS-1 cells, mouse embryos, and various cell lines.