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The Ca2+ pump in rat duodenal epithelium is studied as ATP-dependent Ca2+ uptake in a vesicle preparation with a 9-fold purification in Na+-K+-ATPase activity and a 20-fold purification of Na+-K+-ATPase with respect to an endoplasmic reticulum marker. ATP-dependent Ca2+ uptake is reduced by 60% by digitonin treatment of the vesicles, whereas high-affinity Ca2+-ATPase is stimulated by the same treatment. Different methods to deplete membrane preparations of calmodulin have been used. In EDTA osmotically shocked vesicles, calmodulin stimulated ATP-dependent Ca2+ transport up to 100% in a Ca2+ concentration-dependent manner. The duodenal Ca2+ pump is inhibited by calmodulin antagonists only at low Ca2+ concentrations and in membranes not depleted from calmodulin. Vitamin D-dependent Ca2+-binding protein (Mr = 10,000) in concentrations up to 5 microM did not affect the rate of ATP-dependent Ca2+ transport, either in Ca2+-EGTA-buffered solutions or in EGTA-free solutions. In membrane preparations from vitamin D-deficient rats, the effects of calmodulin and of Ca2+-binding protein were identical to the vitamin D-repleted control preparations. This excludes a specific effect of Ca2+-binding protein and calmodulin in the vitamin D dependency of duodenal Ca2+-ATPase.

The characterization of neuron populations by their immunoreactivity against parvalbumin- and calbindin (28-kDa)-antisera has been used to study the postnatal development of the visual diencephalic nucleus rotundus and the mesencephalic nucleus isthmi complex in zebra finches. In nucleus rotundus, parvalbumin-immunoreactivity was restricted to the neuropil during the first 10 days and appears additionally in somata around day 12 where it remains until adulthood. Calbindin-immunoreactivity of the very scarce neuropil and the few somata, which can be observed during the first two weeks, disappears until adulthood. Thus, the adult nucleus rotundus shows an almost complementary distribution of calbindin- and parvalbumin-immunoreactive structures: the numerous, heavily parvalbumin-positive somata, which are surrounded by dense immunoreactive neuropil are in sharp contrast to the complete absence of calbindin-immunoreactive somata. Only a thin rim surrounding this nucleus contains punctate calbindin-positive neuropil. In the nucleus isthmi complex, parvalbumin and calbindin staining patterns show markedly different developmental profiles. While the density of parvalbumin-immunoreactive neuropil in the parvocellular part of the nucleus isthmi continuously increases and the somata remain unstained, the initially heavily calbindin-positive somata gradually lose their immunoreactivity during the first two weeks. In the adult nucleus isthmi complex, parvalbumin- and calbindin show nearly identical staining patterns. A comparison between the two calcium-binding proteins and GABA-immunoreactivity in adult brains revealed different relationships in the two nuclei: while in nucleus rotundus GABA-staining pattern neither resembles that of parvalbumin nor of calbindin, in the nucleus isthmi complex all three staining patterns coincide.

Parvalbumin, a Ca2+-binding protein, was isolated from rat testis. This is the first demonstration of the protein in endocrine glands. By using a rat parvalbumin cDNA probe, parvalbumin mRNA was demonstrated in the testis, indicating that the protein is synthesized in this tissue and that testis parvalbumin is a product of the same gene as the one encoding for muscle parvalbumin. Parvalbumin was localized by immunohistochemical methods in the Leydig cells and in the acrosome region of maturing spermatids (stages 1-15). The expression of parvalbumin during testis development was followed. High parvalbumin protein and mRNA levels were found at stages of highest Leydig cell activity, i.e. at late fetal stages until birth and again around postnatal day 50. This suggests that parvalbumin may be involved in the production of testosterone in Leydig cells, a process which is highly dependent on calcium.

Calcium and intracellular Ca2+-binding proteins are possibly involved in hormone production and spermatogenesis in rat testis. Parvalbumin, calbindin D-28K, S-100 proteins and calmodulin were localized in the Leydig cells, which are sites of testosterone synthesis. Only the appearance of parvalbumin-immunoreactivity is closely correlated to testosterone production during development of the testes. Calbindin D-28K-immunoreactivity persisted in foetal-type Leydig cells and in adult-type Leydig cells at all stages of development. S-100-immunoreactivity was low during all foetal stages, absent between birth and puberty, and increased thereafter. Calmodulin staining is most prominent in the cytoplasm of developing spermatocytes and of maturing spermatids. All four proteins co-exist in the seminiferous tubules. The distinct localization and developmental appearance of these proteins suggests different regulatory roles in Leydig cell function and spermatogenesis.

Calbindin D-28K-immunoreactive cells were localized in the supragranular layers of the striate cortex of the cat, while parvalbumin-stained cells occurred from the bottom half of layer II through layer VI, making the two distributions almost complementary. Calbindin- and parvalbumin-positive cells occurred throughout the 3 layers of the dorsal lateral geniculate nucleus (dLGN), but calbindin-immunoreactive cells outnumbered parvalbumin-positive cells. Double labeling on single sections was performed in order to determine the possible coexistence of calbindin and parvalbumin in single cells of cat visual cortex and dLGN. Calbindin and parvalbumin immunoreactivity was found in two separate neuronal populations in the visual cortex, while in the dLGN about 50% of the cells were doubly stained.

The type and cell size of parvalbumin-immunoreactive (PV-Ir) neurones were examined in 14 postmortem brains from elderly control and Alzheimer-type dementia (ATD) patients with the aid of an image analyser. Morphological features of PV-Ir neurones suggested the existence of PV in the non-pyramidal interneurones in the cerebral cortex. A significant loss of PV-Ir cells was found in the frontal and temporal cortex in ATD. A significant reduction in the size of PV-Ir cells was also noted in the temporal cortex in ATD. These findings suggested that PV-Ir neurones in the cortex are affected in ATD.

In the cat primary visual cortex, we investigated with immunohistochemical techniques the developmental changes in the cellular and subcellular localization of the Ca2+-binding proteins parvalbumin (PV) and calbindin-D 28K (CBP), in order to determine whether there is a correlation between the expression of Ca2+-dependent processes and the time course of the critical period for use-dependent plasticity. On the 54th day of gestation and at 1 week postnatally, both calcium-binding proteins were present only in a subpopulation of neurons in layers V and VI. During subsequent maturation, the number of PV(+) and CBP(+) neurons increased significantly and labeled cells were detected in more superficial layers. Moreover, the homogeneous labeling of some CBP(+) neurons in layers IV to VI decreased and changed to a punctate pattern. In adult cats PV(+) neurons were evenly distributed throughout layers II to VI, whereas CBP(+) neurons were concentrated in layers II/III. Only a few immunoreactive cells had morphological features characteristic of pyramidal cells; the large majority were nonpyramidal. Electron microscopy confirmed the presence of PV- and CBP-reaction product within the perikarya, axons, and dendrites of labeled cells. It was associated preferentially with microtubules, postsynaptic densities, and intracellular membranes. Immunoreactive neurons received immunonegative asymmetric synapses on their dendritic shafts and made symmetric synaptic contacts with labeled and unlabeled somata and with unlabeled dendritic shafts. The large number and widespread distribution of immunoreactive neurons implies that PV and CBP play an important role in the regulation of calcium-dependent processes in the visual cortex. Furthermore, the developmental redistribution of PV and CBP points to changes in the organization of Ca2+-dependent processes during maturation.

Elevated levels of Ca2+-binding proteins are reported in transformed cells and thought to be involved in their uncontrolled proliferation and often increased motility. Therefore, three cell lines [LICR(Lond)-HN 1, -HN 2, and -HN 6] derived from human carcinomas displaying various degrees of locomotive activity were investigated for the presence of parvalbumin and related Ca2+-binding proteins. By applying different immunohistochemical methods in conjunction with a monospecific anti-parvalbumin antiserum, an intense staining was seen in cells displaying translocative motility. Often in these cells, an association with filamentous structures located in the nuclear region was observed. Unique Ca2+-binding proteins, absent from comparable normal tissue, were found in the malignant cell lines when analyzed by two-dimensional polyacrylamide gel electrophoresis and high-performance liquid chromatography. From these tumor cells a protein (Mr, 12,000; pI, 4.8) was isolated that crossreacted with antiparvalbumin antiserum. Peptide maps of this protein revealed a further structural homology to parvalbumin (Mr, 12,000; pI, 4.9). In analogy to muscle, where there is evidence for a regulatory role of parvalbumin in the contraction-relaxation cycle, we speculate that this protein is tumor-associated and connected to the motile behavior of carcinoma cells.

As part of a study on the evolutionary aspects of control mechanisms, a number of structural muscle components from the Pacific dogfish (Squalus acanthias) are described. These include troponin, tropomyosin, actin, and myosin. Troponin (mol wt 108.000) was resolved into its constitutive subunits, repeated by a 20,500 mol wt fragment which binds 2 mol of Ca2+/mol with a KDiss of 0.91 mum, and an inhibitory component of 30,000 and a 58,000 component which are necessary for the calcium sensitivity of actomyosin ATPase. Tropomyosin and actin share many properties with their counterparts from higher vertebrates. Proteins similar to parvalbumins, i.e., the low molecular weight calcium-binding proteins widely distributed in fish, amphibians, and mammalian muscle, could be generated from troponin and its calcium-binding subunit by limited proteolysis. The appearance of immunological cross-reactivity and other similar features suggested some identity, but differences in the amino acid analysis exclude the possiblity that parvalbumins occur as breakdown products of troponin. The close relationship between parvalbumins and the calcium-binding subunit brings additional evidence that these proteins have arisen through divergent evolution.

Using antibodies against calcium-binding proteins immunocytochemistry revealed quantitative staining of horizontal cells in whole mount preparations. In cat both A- and B-type horizontal cells, and in monkey probable H1- and H2-horizontal cells were labelled with antibodies against parvalbumin. In rabbit and ox two types of horizontal cell were labelled with antibodies against calcium-binding protein (CaBP-28K). In ox retina processes of horizontal cells were observed descending into the inner plexiform layer (IPL).

Parvalbumin, a high affinity Ca2+-binding protein, is known to be expressed only in muscles and brain in the rat. We have investigated its distribution and characteristics in other rat tissues by several biochemical and immunohistochemical methods. Evidence for the presence of parvalbumin in teeth, bone, skin, prostate, seminal vesicles, testes, and ovary is given by two-dimensional polyacrylamide gel electrophoresis, immunoblotting ("Western technique") of one-dimensional gels, and its concentration measured by reverse phase high performance liquid chromatography. The distribution within several parvalbumin-positive organs was monitored by the immunohistochemical peroxidase-antiperoxidase method. In teeth, only ameloblasts reacted with anti-rat parvalbumin serum and in bone the calcified extracellular cartilage was the target of the immunoreaction. The panniculus carnosus was the exclusive site of parvalbumin in the skin. Besides the already known parvalbumin distribution in the brain, parvalbumin is also expressed in distinct cell types of the peripheral nervous system. Leydig cells were found to be the only parvalbumin location in testes. These observations lead us to conclude that parvalbumin in contrast to the multifunctional and constitutive calmodulin must function in Ca2+-dependent processes related to specific cell types.

Antisera against the Ca2+-binding proteins parvalbumin, calbindin D-28K, and the S-100 proteins were used to study the distribution of their target proteins in selected human carcinoma (LICR-HN6;Caco-2), mouse neuroblastoma (clone NB-2a), and rat glioma cell lines (clone C-6). Pronounced staining with anti-parvalbumin was observed in the cytosol of all cells as well as in some nuclei, in particular, mitotic nuclei were highly immuno-reactive. Applying light and immune-electron microscopy (colloidal gold labelling) the parvalbumin-fluorescence was associated with filaments in the LICR-HN6 cells. However, this immunoreactivity was not a result of the presence of parvalbumin itself--as shown by biochemical analyses (HPLC, 2D-PAGE)--but was due to the presence of a Ca2+-binding and tumour-associated protein with similar biochemical and immunological properties. S-100 proteins were present in all tumour cell lines but their intracellular distribution was different from calbindin D-28K. Calbindin-immunoreactivity was found on the membranes of the carcinoma cell lines whereas neuroblastoma and glioma cells remained unlabelled. It is suggested that these proteins might be involved in the modulation of the enhanced stimulation of Ca2+-dependent processes occurring in tumour cells.

A single-stranded DNA-binding protein of Mr 35,000 (35K protein) was isolated from calf cerebral cortex by affinity chromatography on immobilized double-stranded and single-stranded DNA. Its localization in the nuclear compartment was demonstrated by immunohistochemistry. Previous studies had uncovered a homologous nonhistone chromosomal protein in the nuclei of rat cerebral cortex neurons, cerebellar neurons, oligodendrocytes, and liver cells. The rat protein accumulated in the nuclear compartment of neurons in exact temporal coincidence with the arrest of cell division and the initiation of terminal differentiation. Therefore, in the present work, the 35K protein was tested for an activating role in RNA transcription. During the course of this study we became aware that the 35K protein was identical to a glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12). When authentic GAPDH from rabbit skeletal muscle was injected into Xenopus laevis oocytes, it greatly stimulated RNA polymerase II transcription, whereas the 35K protein from calf brain did not. This apparent discrepancy was partially resolved by the finding that rabbit muscle GAPDH could be fractionated into two components by affinity chromatography on single-stranded DNA cellulose. Only 5% of the applied protein was retained on the column and could be eluted with a shallow salt gradient identical to the one used for the isolation of the 35K protein. This single-stranded DNA-binding component of rabbit muscle GAPDH did not stimulate transcription. Apparently, the 35K protein from calf brain corresponded to this single-stranded DNA-binding subfraction, which explained its failure to activate transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuronal parvalbumin has been isolated from rat brain and purified to homogeneity by high-performance liquid chromatography (HPLC) on reverse-phase supports. This procedure includes four consecutive chromatographic steps with an overall protein recovery of 74% and a 26 400-fold purification. The concentration of parvalbumin was found to be approximately 10 mg/kg wet weight in brain tissue, which is about 100 times lower than that in rat muscle. The physical properties of brain parvalbumin are described and compared with those of the muscle counterpart. These proteins were identical in their molecular weights (12 000), isoelectric points (4.9), retention times on C-18 reverse-phase HPLC columns, Ca2+ content (two per molecule), amino acid compositions, and immunological properties. A comparison of the tryptic peptide maps of brain and muscle parvalbumin by analytical HPLC also revealed identity and showed that the isolation method described here did not alter the chemical structure of the protein.

Assuming a possible role of calcium in the function of the germinal proliferation centre in the testis of Drosophila melanogaster, the distribution of Ca2+-binding protein parvalbumin and structural related proteins in male gonads was tested by several biochemical and immunohistochemical methods. The two dimensional PAGE analysis on 3000 gonads of pupal stages suggests the presence of parvalbumin in this tissue. Immunohistochemical studies confirmed this finding. Parvalbumin-immunoreactivity was located in (or near) membraneous systems, like mitochondria and (or) microtubuli. The immunohistochemical analysis of irradiated gonads showed no radiation damage effect on the distribution of parvalbumin in certain cells and cell components.

The fast extensor digitorum longus (EDL) and the slow soleus (SOL) muscles were cross-reinnervated in both directions in the rat. During the following transformation of muscle type properties, the expression of the Ca2+-binding parvalbumin (parvalbumin, Mr = 12,000) was investigated. The combined biochemical, histochemical, and physiological results demonstrated that the amount of parvalbumin decreased in the fast to slow (X-EDL) and increased in the slow to fast (X-SOL) transformation. Alterations of parvalbumin-mRNA levels were similar to changes found at the protein level, indicating a tight transcriptional regulation of the parvalbumin expression. The close correlation, however, between parvalbumin and relaxation speed found in normal muscles had changed after cross-reinnervation. After the altered nervous input, a slow contracting/slow relaxing muscle may even contain more parvalbumin than a fast contracting/fast relaxing one. The expression of parvalbumin may depend on the nerve-muscle interaction, and parvalbumin may thus be used as a sensitive marker for early stages of muscular transformation and neurological disorders.

Skin Ca2+-binding protein (SCaBP) was reported to be distinct from the Ca2+-binding parvalbumin (PV), however, more recently its amino acid sequence was shown to be identical to PV. We purified a protein (Mr 12,000; pI4.5) from isolated epidermis (free of other cell layers) of adult rats and whole skin (containing no PV) of newborn rats. This protein is referred to as epidermal protein (EP-12), distinct from PV in its hydrophobicity, amino acid composition and immunological properties. Previously isolated SCaBP was shown to be a mixture of EP-12 and PV. The localization and possible functions of EP-12 and of PV in skin of adult and newborn rat are discussed.

Calcium ions play a key role in many aspects of neuronal behavior and certain calcium binding proteins that may influence this behavior are differentially distributed in the central nervous system. In this study it is shown that immunoreactivity for calbindin-28 and for parvalbumin is localized in separate populations of inhibitory GABA interneurons in all areas of the neocortex of Old World monkeys. Virtually all GABA neurons show immunoreactivity for one or other calcium binding protein but, except for a few cells in layer IV, GABA cells do not show immunoreactivity for both proteins. Among the two cell populations, parvalbumin immunoreactivity characterizes basket neurons while calbindin immunoreactivity characterizes double bouquet neurons. These findings suggest that the two GABA cell types differ in their regulation of calcium homeostasis and may yield clues to their different roles in intracortical circuitry.

The primary structure of parvalbumin from rat skeletal muscle has been determined principally by automated sequencing of tryptic peptides using 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate as the Edman reagent on a solid-phase sequencer. Remaining positions and most peptide overlaps were identified by analysis of peptides arising from CNBr, chymotryptic and Staphylococcus aureus protease cleavages and through digestions with carboxypeptidases A, B and Y. Reverse-phase high-performance liquid chromatography on C-18 supports was employed for all peptide separations. Structural homology between rat and rabbit parvalbumins helped to confirm the alignments of the tryptic peptides T4-T3, T2-T6 and to define the position of the Lys triplet (36-38). A comparison of the two mammalian proteins revealed 14 amino acid differences, which are all located on the surface of the molecule. A prediction of the secondary structure has been made and found to be very similar for the rat and rabbit proteins with the exception of the sequence region 72-78, located between the Ca2+, Mg2+-binding CD and EF domains.

Two neuronal and Ca2+-binding proteins, parvalbumin and a vitamin-D-dependent calcium-binding protein (CaBP; Mr = 28,000) were localized immunohistochemically in the rat cerebellum, which is the brain region containing the highest concentration of them both. Parvalbumin was present in Purkinje, basket and stellate cells of the molecular layer, whereas CaBP was only present in Purkinje cells. The concentrations of both proteins, measured immunohistochemically and biochemically, were unaffected by the vitamin D status of the rats.

Parvalbumin was isolated from human cerebral cortex and biceps and triceps muscles by HPLC. The immunological properties of the human protein and the mobility in two-dimensional polyacrylamide gels were similar to that of parvalbumin isolated from the muscles of rat, mouse, rabbit, and chicken. The tryptic peptide maps of the human parvalbumin, however, differed considerably from all other parvalbumins, indicating a distinct primary structure. The immunolabeled cells in the hippocampus of the human brain were of different sizes and forms; they occurred in all subfields and probably represent interneurons.

The physiological role of the Ca2+-binding protein parvalbumin in skeletal muscle has been investigated by measuring the parvalbumin content by HPLC in a variety of mammalian muscles, including man, and comparing the results with the respective muscle relaxation properties and fiber type compositions. The parvalbumin concentrations were highest in the skeletal muscles of the smallest animal investigated (mouse, gastrocnemius: 4.9 g/kg), which has the highest relaxation speed, and lowest in the larger animals (horse, deep gluteal muscle: less than or equal to 0.001 g/kg) and man (vastus, triceps: less than or equal to 0.001 g/kg), which have much lower relaxation speeds. Analysis of three type-homogeneous muscles of the guinea pig revealed highest parvalbumin concentrations (0.25 g/kg) in sartorius (type IIB) and lowest concentrations (less than or equal to 0.007 g/kg) in soleus (type I), consistent with the different half-relaxation times of fast and slow muscles. Denervation of the rat extensor digitorum longus, which increases the half-relaxation time from 9.4 to 19 msec, resulted in a 20% decrease of the parvalbumin content. Given this close correlation between parvalbumin content and relaxation speed in a variety of muscles and species, we suggest that parvalbumin is involved directly in the relaxation process in fast muscles.

Lectins with specific affinity for terminal N-acetylgalactosamine, Vicia villosa agglutinin (VVA) and Glycine max agglutinin (soybean agglutinin; SBA), are shown to stain selectively a subpopulation of GABAergic neurons in the rat cerebral cortex. About 90% of VAA- and/or SBA-stained cells are also parvalbumin-immunoreactive, but no VVA-stained cells showed somatostatin-28-like or cholecystokinin-8-like immunoreactivities.

The structure of the rat parvalbumin gene has been elucidated from analysis of six overlapping clones isolated from a rat lambda Charon 4A genomic library. Two of the clones were mapped in detail, and all exons were localized by Southern hybridization using fragments of a full-length parvalbumin cDNA (Epstein, P., Means, A. R., and Berchtold, M. W. (1986) J. Biol. Chem. 261, 5886-5891). The rat parvalbumin transcription unit is 15.5 kilobase pairs in length and contains four introns. The first intron divides the 5'-nontranslated region, whereas the other three interrupt coding DNA. All intron/extron boundaries were sequenced as was 377 base pairs immediately 5' from the putative transcription initiation site. The promoter region contains eukaryotic regulatory homologies to the "TATA" box at -24 and "CAAT" box at -47 and -156. In addition, two doublets consisting of 11-base pair direct repeats exist in the promoter region. Parvalbumin binds two Ca2+, whereas many other members of the same superfamily bind four. Comparison of the genes that encode these proteins provides a strong confirmation of the hypothesis that parvalbumin evolved from an ancestral gene specifying a four-domain Ca2+-binding protein. The rat parvalbumin gene was also utilized to assign its human counterpart to chromosome 22 from data obtained by hybridization to DNA from a somatic cell hybrid panel. It was also used to isolate a 7.5-kilobase pair EcoRI fragment from a human chromosome 22 DNA library.

Antibodies to parvalbumin label intensely a small number of non-overlapping fibre systems in embryonic rat brain. All are in hindbrain--the oculomotor and trochlear motor fibres, the acoustic and vestibular fibres of the VIIIth nerve, and an unidentified group of fibres which ascend under the dorsal surface in caudal medulla. Of these, the vestibular fibres are the first to acquire parvalbumin immunoreactivity, and we have used this property to follow the growth of their axons in the brain. This occurs in two phases. In the first, occurring at embryonic days 12-14, the axons grow in small groups or fascicles under the pial surface to their most distant terminal zones rostrally in the cerebellum and caudally in the descending vestibular nuclei. This growth is directed towards the two sites where germinal neuroepithelium is expanding over the medullary velum in forming the cerebellum and lateral recess of the IVth ventricle. In a second stage, commencing at E15, individual collaterals branch from these fascicles to arborize amongst their presumptive synaptic targets (cells of the vestibular nuclei and vestibulocerebellum) located in the sub-ventricular and ventricular layers. In this phase the axons follow a radial route, at right angles to their original subpial course, possibly by growing along radial glial processes. The target cells then migrate to their final position with the vestibular axons maintaining contact with them. The vestibular fibres are the first axons to enter the cerebellum, but from E15 onwards their fascicles are joined by increasing numbers of non-vestibular fibres following the same course. These other axons, and the movement of cells to form the deep cerebellar nuclei, separate the fascicles of vestibular fibres so that their course into the cerebellum becomes very diffuse. Thus this single set of axons grow, not only in two distinct phases, but also follow distinctly different substrates for growth in each. Furthermore, they then appear to act as pioneer fibres guiding the entry or egress of later-developing axons to or from the cerebellum.

The localization of several muscle proteins in relaxed and contracted myofibrils is compared. The morphology of the myofibrils and the behavior of these proteins was also investigated under extraction conditions.

Gap junctions were identified for the first time on chemically defined neurons in the central nervous system. Gap junctions were thus demonstrated on GABAergic neurons containing the calcium-binding on GABAergic neurons containing the calcium-binding protein parvalbumin (PV) in the rat hippocampus. Thin and semithin (0.5 micron thick) sections were cut alternately and consecutively from osmium-fixed tissue which was embedded in epoxy resin and usable for conventional electron microscopic studies. The semithin sections were processed for postembedding immunocytochemistry using an anti-PV serum. Structures corresponding to the PV-immunoreactive (PV-I) profiles on the semithin sections were easily identified on electron micrographs from the adjacent thin sections. Using this technique gap junctions were found (1) between PV-I dendrites, (2) between PV-I dendrites and PV-I somata and (3) between PV-I dendrites and small processes whose origin could not be identified. Despite a systematic search, we did not find gap junction between PV-negative processes.

The coexistence in individual neurons of parvalbumin and gamma-aminobutyric acid (GABA) was studied in the dorsal lateral geniculate nucleus (dLGN) of the cat using pre- and postembedding immunocytochemical methods. PV(+) cell bodies and processes were found in the perigeniculate nucleus (PGN) and throughout all laminae of the dLGN. PV(+) neurons were relatively small and had circular to fusiform shapes. Electron microscopy revealed PV(+) reaction product within the perikarya, axons, and dendrites of labeled cells. It was associated preferentially with microtubules, postsynaptic densities, and intracellular membranes. PV(+) presynaptic boutons were identified on the basis of their synaptic relations and ultrastructure as retinal terminals (RLP) and as profiles originating from inhibitory interneurons (F1 and F2). Immunopositive somata and dendrites received asymmetric synaptic contacts from labeled RLP and non-identified, non-immunoreactive synaptic boutons. Moreover, PV(+) dendrites were postsynaptic to labeled F profiles. In the PGN all neurons were both PV(+) and GABA-immunoreactive and in the dLGN the vast majority of PV(+) neurons showed GABA-immunoreactivity. It is suggested that the high incidence of PV in GABAergic neurons is related to the particular activation patterns of these neurons and the resulting demand for calcium buffer systems.

The structural features of PV-immunoreactive (PV-I) neurons, a particular subpopulation of GABAergic neurons, in the hippocampus were studied by immunocytochemistry. The PV-I cell bodies were concentrated within the stratum pyramidale (SP) and stratum oriens (SO) in the hippocampus. PV-I puncta were frequent in SP, while they were rarely seen in other layers. The dendritic arborization of PV-I neurons resembled that of some of the nonpyramidal cells observed after Golgi-impregnation. The most commonly observed PV-I neurons had their perikarya located in SP with dendrites extending into SO and the stratum radiatum (SR). Most of the dendrites in SR had typical beaded or varicose segments. The dendrites extending into SO had few beaded parts. There were many bipolar and multipolar neurons with smooth dendrites in SO, but only a small number of such multipolar neurons in SR. An electron microscopic analysis revealed that PV-I products were located to perikarya, dendrites, myelinated axons and synaptic boutons. The perikarya of PV-I neurons exhibited several ultrastructural features of nonpyramidal cells, e.g., abundant cisternae of endoplasmic reticulum, mitochondria and other perikaryal organelles, an infolded nuclear envelope and intranuclear inclusions. They received many asymmetric synapses with round presynaptic vesicles. There were numerous PV-I boutons, presumably axonal endings, covering the pyramidal cell bodies. The PV-I boutons also contacted the axon initial segments and proximal dendrites of the pyramidal cells. In addition PV-I terminals were found on somata and dendrites of both PV-I or PV-negative nonpyramidal cells. The results suggest that PV-containing neurons include basket and axo-axonic cells.

Calcium, acting as second messenger, are through-target proteins that contain an EF-hand structure. Based on this general principle, EF-hand structures can be predicted from the amino acid sequence of a protein. Most recently, several new members of the Ca2+-binding protein family have been discovered, e.g., the cystic fibrosis antigen (CFAg), the macrophage migration inhibitory factor related proteins (MRP-8 and -14), non-muscle alpha-actinin, and uvomorulin, the latter belonging to the group of cell adhesion molecules. Additionally, new information about the physiological functions of S-100 proteins, calbindins, calretinin as well as parvalbumin were observed and some of the these results are discussed.

The Ca2+-binding parvalbumin (PV) is possibly involved in the relaxation of fast-twitch muscle fibers and believed to be a marker for early muscular disturbances. The muscular content of parvalbumin has been shown to change with alterations of the relaxation speed that follow an experimentally changed nervous input. In hypo- and hyperthyroidism isometric twitch contraction and half-relaxation times are also altered, namely increased in hypothyroidism and decreased in hyperthyroidism. These changes are largely paralleled by modifications in the fiber type composition. Therefore we investigated the distribution and concentration of parvalbumin in extensor digitorum longus, soleus, and gastrocnemius muscles of rats by immunohistochemical and biochemical methods. The combined results of both procedures showed that parvalbumin distribution and concentration were largely unaffected in all thyroid states. This suggests that the expression of parvalbumin is neuronally controlled and not by thyroid hormones. Additionally our findings support the view that the changes in physiologic properties and fiber type composition are generated by a direct action of thyroid hormone on muscle fibers, and not via their nervous input.

Reverse-phase high-performance liquid chromatography has been shown to be applicable to the isolation of Ca2+-binding proteins, specifically parvalbumins, from tissue extracts or from preparations first purified by "conventional" chromatography. Through an investigation of the behavior of a series of Ca2+-binding proteins as a function of buffer composition, pH, and organic eluant it has been possible to define mild conditions allowing for chromatography of the proteins in their native states. The elution positions of parvalbumins were not observed to correlate with the "overall" protein hydrophobicity, calculated using hydrophobicity values for the individual amino acids, thus indicating that factors such as hydrophobic/hydrophilic surface areas are important in determining the degree of association with the support. The usefulness of reverse-phase chromatography as an analytical tool for determining protein homogeneity is illustrated. Samples which had been isolated via "conventional" chromatography methods, and thought to be homogeneous, were observed to contain multiple species of the same protein.

The structure of a novel long terminal repeat (LTR) from an intracisternal A particle (IAP) DNA element in the rat (Sprague-Dawley) genome was determined. This LTR has a total length of 313 base pairs (bp). Several structural features typical for retroviral LTR promoters were identified, including a "CCAAT" box, a "TATA" box, a polyadenylation signal, and a polyadenylation site. The LTR is flanked by 3-bp inverted repeats, and it consists of the three typical LTR regions, U3, R, and U5. U3 contains 213 bp, R 46 bp, and U5 54 bp, which is within the usual size range of IAP LTRs. A sequence of 60 bp in the U3 region reveals considerable similarity to a murine IAP LTR U3 element, which is known to interact with nuclear proteins. A sequence of 69 bp in the U5 and R regions has 83 and 93% similarities to an endogenous retroviral LTR from Syrian hamster and to the cDNA leader sequence of (Buffalo) rat oncomodulin, respectively. Oncomodulin is an "EF-hand" Ca2+-binding protein and appears in many human and rodent tumors and in cells with tumor-like properties but not in normal tissues. We postulate that in the rat the tumor-specific expression of oncomodulin is controlled by a retroviral LTR promoter.