Selective immunocytochemical localisation of calretinin in the human ovary

 Calretinin is a calcium-binding protein which is primarily expressed by certain cells of the nervous system, both central and peripheral. Its presence in non-excitable cells has been little studied. Using a polyclonal antibody and paraffin sections we have analysed the presence of calretinin in the human ovary of different ages, and in polycystic ovaries. Our results revealed the selective presence of calretinin, specifically localised in the cells of the germinal epithelium, those of the theca interna and the theca lutein cells, in the cells of the neuro-vaso-epithelial association and of the canalicular structures of the mesovarium. Calretinin was also present in a few cells of the theca externa and some interstitial cells. No appreciable quantitative differences in the strength of the positive reaction were seen between the ovaries of different ages or between the normal and the polycystic ovaries. The presence of calretinin was confirmed by western blot analysis. The selective presence of calretinin in the human ovary, in androgenic cells and in the cells of the germinal epithelium is discussed in relation to its possible function as a Ca²⁺ buffer. Accepted: 2 June 1997     

Nucleotide sequence of rat calretinin cDNA

A rat calretinin cDNA clone was selected by antibody screening of a λgt11 brain library. The sequence revealed remarkable nucleotide and amino acid homology with human calretinin (91.1% and 98.5%, respectively), with only four amino acid differences. A high degree of homology with chick calretinin was also observed (79.8% and 86.6%, respectively), with 36 amino acid differences.

Although the role of this central nervous system protein has not been well characterized, the evolutionarily conserved calcium binding domains and connecting regions, in addition to the limited local changes observed between rat and chick primary structure, lead us to believe that calretinin interacts with other highly conserved constituents of brain cells. This calretinin cDNA clone provides a new probe for the analysis of a specific subset of neurons in the central nervous system. The probe will allow a more detailed analysis of calretinin regulation in the brain and will be useful for screening genomic libraries for the complete chromosomal gene. (GenBank accession No. X66974.)     

Quadruple colocalization of calretinin,calcitonin gene-related peptide,vasoactive intestinal peptide,and substance P in fibers within the villi of the rat intestine

Double-labeling immunofluoresenct histochemistry demonstrates that calretinin, a calcium-binding protein, coexists with calcitonin gene-related peptide, vasoactive intestinal peptide, and substance P in the fibers innervating the lamina propria of the rat intestinal villi. An acetylcholinesterase histochemical stain revealed that the majority of calretinin-containing cells in the myenteric ganglia were cholinergic and that about one half of the submucosal calretinin-containing cells colocalized with acetylcholinesterase. In situ hybridization studies confirmed the presence of calretinin mRNA in the dorsal root ganglia, and a ribonuclease protection assay verified the presence of calretinin message in the intestine. The coexistence of calretinin in calcitonin-gene-related-peptide-containing cells that also contained substance P and vasoactive intestinal polypeptide in the dorsal root ganglia suggest that these ganglia are the source of the quadruple colocalization within the sensory fibers of the villi. Although the function of calretinin in these nerves is unknown, it is hypothesized that the coexistence of three potent vasodilatory peptides influences the uptake of metabolized food products within the vasculature of the villi.     

Calretinin and calbindin in the retina of the developing chick

Summary Calretinin and calbindin-D28k are two calcium-binding proteins that are present in largely different sets of nerve cells in the central nervous system. Their appearance during development of the chick retina was studied by immunohistochemistry and Western blots. The patterns are mature one day before hatching. Each cell type acquires its characteristic calcium-binding protein several days after its differentiation has started, but in most cases before morphological maturation is complete. There is also an early phase of calbindin immunoreactivity in many immature amacrine cells, and of calretinin immunoreactivity in the presumptive photoreceptor layer, suggesting that these proteins may have distinct functions in differentiating cells.Abbreviations CR+ Immunoreactive for calretinin only - CB+ immunoreactive for calbindin only - CR+CB+ immunoreactive for both antisera - IPL inner plexiform layer - OPL outer plexiform layer     

The value of calretinin and cytokeratin 5/6 as markers for mesothelioma in cell block preparations of serous effusions

Objective: To determine the value of calretinin and cytokeratin (CK) 5/6 in discriminating mesothelioma from adenocarcinoma in serous effusion specimens. Methods: A total of 101 recent, histologically or clinically confirmed malignant effusions with immunostained cell block preparations were reviewed. The cases consisted of 34 mesotheliomas and 67 adenocarcinomas. This included 17 ascitic fluid and 84 pleural fluid samples. The adenocarcinomas included metastatic carcinomas from the breast (12), lung (19), stomach (3), colon (1), pancreas (2), ovary (6) endometrium (1) and 23 histologically confirmed metastases from unknown primary sites. The cases were assessed as negative or positive (>5% of cells stained). The staining pattern was recorded as cytoplasmic, cell membrane, nuclear or cytoplasmic and nuclear staining. Results: Calretinin staining was present in 97% (33/34) of the mesothelioma cases with a majority of them showing both cytoplasmic and nuclear staining (29/33). Only 3% (2/67) of adenocarcinomas were positive for calretinin, one being a lung adenocarcinoma and the other an adenocarcinoma of unknown primary site in an ascitic fluid. Cytokeratin 5/6 staining was also present in 33/34 (97%) of mesothelioma cases. Six (9%) adenocarcinomas were positive, including metastases from the lung (1), breast (1), ovary (2) and unknown primary site (2). Four of the six adenocarcinoma cases positive for CK5/6 were in ascitic fluids. No cases of mesothelioma were negative for both calretinin and CK5/6. Only one adenocarcinoma case, (which was from unknown primary site in an ascitic fluid sample), was positive for both markers. Conclusions: The results confirm that calretinin and CK 5/6 are useful markers for mesothelioma in effusion specimens. CK5/6 staining may be less useful for peritoneal fluid specimens where metastatic adenocarcinomas may be more likely to express the antigen. Further study of ascitic/peritoneal specimens is warranted. However, positive staining, particularly for both antigens, is highly indicative of a mesothelial origin for cells. The two markers make a useful addition to EMA and the panel of adenocarcinoma markers routinely applied to effusion specimens.     

Expression of Calcium-Binding Proteins in Layer 1 Reelin-Immunoreactive Cells during Rat and Mouse Neocortical Development

Cajal-Retzius cells in layer 1 of the developing cerebral cortex and their product of secretion, reelin, an extracellular matrix protein, play a crucial role in establishing the correct lamination pattern in this tissue. As many studies into reelin signaling routes and pathological alterations are conducted in murine models, we used double-labeling and confocal microscopy to compare the distribution of the cell-specific markers, calretinin and calbindin, in reelin-immunoreactive cells during postnatal rat and mouse neocortical development. In the rat, neither calretinin nor calbindin colocalized with reelin in Cajal-Retzius cells at P0-P2. From P5 to P14, the colocalization of reelin and calretinin was commonly found in presumptive rat subpial piriform cells. These cells progressively lacked calretinin expression and persisted into adulthood as part of the pool of layer 1 reelin-positive interneurons. Conversely, in the mouse, reelin-immunoreactive Cajal-Retzius cells colocalized with calretinin and/or calbindin. Subpial piriform cells containing reelin and calretinin were identified at P5-P7, but lacked calretinin expression at P14. In adult mice, as in the rat, reelin-immunoreactive cells did not colocalize with calcium-binding proteins. Our results reveal a complex neurochemical profile of layer 1 cells in the rat neocortex, which makes using a single calcium-binding protein as a marker of rodent reelin-immunoreactive cells difficult.     

Calretinin: Modulator of neuronal excitability

Calretinin is a member of the calcium-binding protein EF-hand family first identified in the retina. As with the other 200-plus calcium-binding proteins, calretinin serves a range of cellular functions including intracellular calcium buffering, messenger targeting, and is involved in processes such as cell cycle arrest, and apoptosis. Calcium-binding proteins including calretinin are expressed differentially in neuronal subpopulations throughout the vertebrate and invertebrate nervous system and their expression has been used to selectively target specific cell types and isolate neuronal networks. More recent experiments have revealed that calretinin plays a crucial role in the modulation of intrinsic neuronal excitability and the induction of long-term potentiation (LTP). Furthermore, selective knockout of calretinin in mice produces disturbances of motor coordination and suggests a putative role for calretinin in the maintenance of calcium dynamics underlying motor adaptation.     

Comparative studies of calretinin expression by WiDr cell line in vivo in xenografts in nude mice and in vitro.

WiDr cells from a human colon adenocarcinoma cultivated in vitro express the calcium binding protein calretinin. The immunoreactivity is present in some interphasic cells and decreases after seven days in culture together with the augmentation of the cell number. Calretinin expression is maintained in the undifferentiated cells of the tumoral mass developed in nude mice and in recultivated isolated tumour cells from the xenograft. From the experiments here described, the protein expression is quantitatively influenced in vitro by the addition of drugs, such as colchicine and taxol, which intervene in cytoskeleton organisation. The percentage of the calretinin immunoreactive cells increases after the addition of colchicine to the medium while the immunoblot analysis shows a higher calretinin content in the cells treated with taxol.     

Calretinin is expressed in the Leydig cells of rat testis

Calretinin, a highly evolutionarily conserved E-F hand calcium binding protein, is expressed predominantly in neurons, with a few exceptions. The function of calretinin is not known. We demonstrate the expression of calretinin mRNA and protein in rat testes. Immunocytochemistry and in situ hybridization reveal that calretinin expression in testis is localized to the interstitial Leydig cells. Western blot and ribonuclease protection analyses show that calretinin protein and mRNA in testis is the same as that expressed in brain. It is suggested that calretinin may play a role in the production of testosterone.     

Expression of Notch1 and Jagged2 in the enteric nervous system.

The Notch signaling pathway is a vitally important pathway in regulating brain development. To explore the involvement of the Notch pathway in neuronal cells of adult rat gut, we investigated the expression of Notch1 and Jagged2 by in situ hybridization (ISH) and immunohistochemistry (IHC). In the enteric nervous system, Notch1 and Jagged2 were expressed in ganglia of the submucosal and myenteric plexus. Notch1 was preferentially expressed in cholinergic neurons lacking calretinin or nitric oxide synthase (NOS), whereas Jagged2 was present in most neuron subtypes. We propose that Notch1 and Jagged2 have a continuing role in the maintenance and function of neuronal cells in the adult enteric nervous system.     

Immunocytochemical localization of nitric oxide synthase-containing neurons in mouse and rabbit visual cortex and co-localization with calcium-binding proteins

Nitric oxide (NO) occurs in various types of cells in the central nervous system. We studied the distribution and morphology of neuronal nitric oxide synthase (NOS)-containing neurons in the visual cortex of mouse and rabbit with antibody immunocytochemistry. We also compared this labeling to that of calbindin D28K, calretinin, and parvalbumin. Staining for NOS was seen both in the specific layers and in selective cell types. The densest concentration of intense anti-NOS immunoreactive (IR) neurons was found in layer VI, while the weak anti-NOS-IR neurons were found in layer II/III in both animals. The NOS-IR neurons varied in morphology. The large majority of NOS-IR neurons were round or oval cells with many dendrites coursing in all directions. Two-color immunofluorescence revealed that only 16.7% of the NOS-IR cells were double-labeled with calbindin D28K in the mouse visual cortex, while more than half (51.7%) of the NOS-IR cells were double-labeled with calretinin and 25.0% of the NOS-IR cells were double-labeled with parvalbumin in mouse. By contrast, 92.4% of the NOS-IR neurons expressed calbindin D28K while only 2.5% of the NOS-IR neurons expressed calretinin in the rabbit visual cortex. In contrast with the mouse, none of the NOS-IR cells in the rabbit visual cortex were double-labeled with parvalbumin. The results indicate that neurons in the visual cortex of both animals express NOS in specific layers and cell types, which do not correlate with the expression of calbindin D28K, calretinin or parvalbumin between the two animals.     

Mouse very long-chain Acyl-CoA synthetase 3/fatty acid transport protein 3 catalyzes fatty acid activation but not fatty acid transport in MA-10 cells

The family of proteins that includes very long-chain acyl-CoA synthetases (ACSVL) consists of six members. These enzymes have also been designated fatty acid transport proteins. We cloned full-length mouse Acsvl3 cDNA and characterized its protein product ACSVL3/fatty acid transport protein 3. The predicted amino acid sequence contains two highly conserved motifs characteristic of acyl-CoA synthetases. Northern blot analysis revealed that the mouse Acsvl3 mRNA is highly expressed in adrenal gland, testis, and ovary, with lower expression in the brain of adult mice. A developmental Northern blot revealed that Acsvl3 mRNA levels were significantly higher in embryonic mouse brain (embryonic days 12-14) than in newborn or adult mice, suggesting a possible role in nervous system development. Immunohistochemistry revealed high ACSVL3 expression in adrenal cortical cells, spermatocytes and interstitial cells of the testis, theca cells of the ovary, cerebral cortical neurons, and cerebellar Purkinje cells. Endogenous ACSVL3 was found primarily in mitochondria of MA-10 and Neuro2a cells by both Western blot analysis of subcellular fractions and immunofluorescence analysis. In MA-10 cells, loss-of-function studies using RNA interference confirmed that endogenous ACSVL3 is an acyl-CoA synthetase capable of activating both long-chain (C16:0) and very long-chain (C24:0) fatty acids. However, despite decreased acyl-CoA synthetase activity, initial rates of fatty acid uptake were unaffected by knockdown of Acsvl3 expression in MA-10 cells. These studies cast doubt on the designation of ACSVL3 as a fatty acid transport protein.     

Inhibition of the Proliferative Cycle and Apoptotic Events in WiDr Cells after Down-Regulation of the Calcium-Binding Protein Calretinin Using Antisense Oligodeoxynucleotides

The colon adenocarcinoma cell line WiDr expresses the calcium-binding protein calretinin (CR). In order to deduce possible functions of calretinin in these cells we decreased its concentration by antisense techniques. Treatment of WiDr cells with phosphorothioate antisense oligodeoxynucleotides (AS-ODNs) led to a drop in calretinin expression, as evidenced by immunohistochemical staining of WiDr cells and Western blot analysis of cytosolic cell extracts. The morphology of these epithelial cells changed from polygonal to spherical and they formed dense cell clusters. Cells displaying morphological alterations typical for apoptotic cells were observed after incubation with AS-ODNs, as evidenced by phase-contrast and electron microscopy. The mitotic rate of AS-ODN-treated cells dropped significantly, as demonstrated by mitotic labeling and time-lapse microcinematography. Furthermore, an accumulation of cells in phase G1 and a reduction of [³H]thymidine-labeled cells was observed in antisense-treated cells. The basal level of [Ca²⁺]_iwas not influenced by the down-regulation of calretinin. WiDr cells incubated with the nonsense, reverse-sense, or with an oligodeoxynucleotide with a totally unrelated sequence did not show any significant differences when compared to control cells. We conclude that calretinin levels have an impact on the progression of the cell cycle of WiDr cells.     

Expression of the calcium binding protein calretinin in WiDr cells and its correlation to their cell cycle

Ca2+ ions intervene during different phases of the progression of the cell cycle, but only one calcium-binding protein, calmodulin, has been shown to be associated with dividing cells. We therefore screened cancer cells for the presence of other related calcium-binding proteins. Using molecular biological and immunohistochemical techniques we show that human tumor cells of epithelial origin, express calretinin. Calretinin immunoreactivity can be demonstrated at precise moments of the cell cycle and, in particular, in phase G1 and during mitosis. During mitosis calretinin is localized both in the cytoplasm and in the mitotic spindle. In the cytoplasm we find calretinin after prophase and until telophase. In the spindle apparatus, calretinin is already present in cells in prometaphase and persists in all the succeeding mitotic phases. It is associated with the kinetochore microtubules but, in contrast to calmodulin, also with the polar microtubules. The role that calretinin plays in well-defined moments of the cell cycle of these cells is as yet unknown, but our results strongly suggest that, in collaboration with other molecules, calretinin intervenes in the dynamic phenomena regulating the separation of the chromosomes.     

Structure of the human brain calcium-binding protein calretinin and its expression in bacteria

Calbindin D28k and calretinin are two closely related intracellular calcium-binding proteins belonging to the troponin C superfamily. Calbindin is known to be involved in the vitamin-D-dependent calcium absorption through intestinal and renal epithelia, while the function of neuronal calbindin and calretinin is poorly understood. Using antibodies directed against chick intestinal calbindin D28k, human calretinin cDNA clones were isolated from brain cDNA libraries. The sequence of the calretinin cDNA revealed an open reading frame of 271 codons coding for a protein of 31,520 Da, and sharing 58% identical residues with human calbindin D28k. Calretinin contains five presumably active and one presumably inactive calcium-binding domains. Comparison with the partial sequences available for chick and guinea pig calretinins revealed that the protein is highly conserved in evolution (evolutionary rate: 0.27 x 10(-9) amino acid-1 year-1). The calretinin message was detected in the brain, while absent from heart muscle, kidney, liver, lung, spleen, stomach and thyroid gland. Recombinant calretinin was expressed in Escherichia coli, and the calcium-binding properties were confirmed on both the natural and the recombinant proteins. Part of the human gene coding for calretinin was isolated and the region corresponding to the promoter and the first exon was sequenced.     

Calretinin immunoreactivity in cholinergic motor neurones,interneurones and vasomotor neurones in the guinea-pig small intestine

Summary Immunoreactivity for calretinin, a calcium-binding protein, was studied in neurones in the guinea-pig small intestine. 26±1% of myenteric neurones and 12±3% of submucous neurones were immunoreactive for calretinin. All calretinin-immunoreactive neurones were also immunoreactive for choline acetyltransferase and hence are likely to be cholinergic. In the myenteric plexus, two subtypes of Dogiel type-I calretinin-immunoreactive neurones could be distinguished from their projections and neurochemical coding. Some calretinin-immunoreactive myenteric neurones had short projections to the tertiary plexus, and hence are likely to be cholinergic motor neurones to the longitudinal muscle. Some of these cells were also immunoreactive for substance P. The remaining myenteric neurones, immunoreactive for calretinin, enkephalin, neurofilament protein triplet and substance P, are likely to be orad-projecting, cholinergic interneurones. Calretinin immunoreactivity was also found in cholinergic neurones in the submucosa, which project to the submucosal vasculature and mucosal glands, and which are likely to mediate vasodilation. Thus, calretinin immunoreactivity in the guinea-pig small intestine is confined to three functional classes of cholinergic neurones. It is possible, for the first time, to distinguish these classes of cells from other enteric neurones.     

Movement of calmodulin between cells in the ovary and embryo of drosophila

Calmodulin (CaM) is an essential component of calcium signaling in multicellular organisms. We used null mutations of the Drosophila CaM gene (Cam) in combination with clonal analysis and immunolocalization to examine the effects of loss of Cam function in the ovarian germline and developing embryo. These studies have uncovered unexpected and striking movements of CaM protein within these tissues. In the ovary, evidence for transfer of CaM from an external source, across plasma membranes, into the germline cells was obtained. In late embryogenesis, maternally derived CaM protein relocalizes dramatically within the nervous system of both wildtype and Cam null embryos-a process that may also involve movement across cell membranes. These findings indicate dynamic, unsuspected elements to the in vivo functions of CaM in the whole organism.     

GFAP transgenic zebrafish

We have generated transgenic zebrafish that express green fluorescent protein (GFP) in glial cells driven by the zebrafish glial fibrillary acidic protein (GFAP) regulatory elements. Transgenic lines Tg(gfap:GFP) were generated from three founders; the results presented here are from the mi2001 line. GFP expression was first visible in the living embryo at the tail bud-stage, then in the developing brain by the 5-somite-stage ( approximately 12 h post-fertilization, hpf) and then spreading posteriorly along the developing spinal cord by the 12-somite stage (approximately 15 hpf). At 24 hpf GFP-expressing cells were in the retina and lens. By 72 hpf GFP expression levels were strong and localized to the glia of the brain, neural retina, spinal cord, and ventral spinal nerves, with moderate expression in the enteric nervous system and weaker levels in the olfactory sensory placode and otic capsule. GFP expression in glia co-localized with anti-GFAP antibodies, but did not co-localize with the neuronal antibodies HuC/D or calretinin in mature neurons.     

Isolation of Enteric Nervous System Progenitor Cells from the Aganglionic Gut of Patients with Hirschsprung’s Disease

Enteric nervous system progenitor cells isolated from postnatal human gut and cultured as neurospheres can then be transplanted into aganglionic gut to restore normal patterns of contractility. These progenitor cells may be of future use to treat patients with Hirschprung’s disease, a congenital condition characterized by hindgut dysmotility due to the lack of enteric nervous system ganglia. Here we demonstrate that progenitor cells can also be isolated from aganglionic gut removed during corrective surgery for Hirschsprung’s disease. Although the enteric nervous system marker calretinin is not expressed in the aganglionic gut region, de novo expression is initiated in cultured neurosphere cells isolated from aganglionic Hirschsprung bowel. Furthermore, expression of the neural markers NOS, VIP and GFAP also increased during culture of aganglionic gut neurospheres which we show can be transplantation into cultured embryonic mouse gut explants to restore a normal frequency of contractility. To determine the origin of the progenitor cells in aganglionic region, we used fluorescence-activated cell sorting to demonstrate that only p75-positive neural crest-derived cells present in the thickened nerve trunks characteristic of the aganglionic region of Hirschsprung gut gave rise to neurons in culture. The derivation of enteric nervous system progenitors in the aganglionic gut region of Hirschprung’s patients not only means that this tissue is a potential source of cells for future autologous transplantation, but it also raises the possibility of inducing the differentiation of these endogenous cells in situ to compensate for the aganglionosis.