Preservation of VGLUT1 synapses on ventral calbindin-immunoreactive interneurons and normal locomotor function in a mouse model of spinal muscular atrophy.

Dysfunction in sensorimotor synapses is one of the earliest pathological changes observed in a mouse model [spinal muscular atrophy (SMA)Δ7] of spinal muscular atrophy. Here, we examined the density of proprioceptive and cholinergic synapses on calbindin-immunoreactive interneurons ventral to the lateral motor column. This population includes inhibitory Renshaw interneurons that are known to receive synaptic input from muscle spindle afferents and from motoneurons. At postnatal day (P)13, near the end stage of the disease, the somatic area of calbindin(+) neurons in the L1/L2 and L5/L6 segments was reduced in SMAΔ7 mice compared with controls. In addition, the number and density of terminals expressing the glutamate vesicular transporter (VGLUT1) and the vesicular acetylcholine transporter (VAChT) were increased on calbindin(+) cells in the L1-L2 but not in the L5-L6 segments of SMAΔ7 mice. In addition, the isolated spinal cord of SMA mice was able to generate locomotor-like activity at P4-P6 in the presence of a drug cocktail or in response to dorsal root stimulation. These results argue against a generalized loss of proprioceptive input to spinal circuits in SMA and suggest that the loss of proprioceptive synapses on motoneurons may be secondary to motoneuron pathology. The increased number of VGLUT1(+) and VAChT(+) synapses on calbindin(+) neurons in the L1/L2 segments may be the result of homeostatic mechanisms. Finally, we have shown that abnormal locomotor network function is unlikely to account for the motor deficits observed in SMA mice at P4-6.

An in-cell NMR study of monitoring stress-induced increase of cytosolic Ca2+ concentration in HeLa cells.

Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. The lifetime of in-cell NMR samples is however much shorter than that in culture media, presumably because of various stresses as well as the nutrient depletion in the anaerobic environment within the NMR tube. It is well known that Ca(2+)-bursts occur in HeLa cells under various stresses, hence the cytosolic Ca(2+) concentration can be regarded as a good indicator of the healthiness of cells in NMR tubes. In this study, aiming at monitoring the states of proteins resulting from the change of cytosolic Ca(2+) concentration during experiments, human calbindin D9k (P47M+C80) was used as the model protein and cultured HeLa cells as host cells. Time-resolved measurements of 2D (1)H-(15)N SOFAST-HMQC experiments of calbindin D9k (P47M+C80) in HeLa cells showed time-dependent changes in the cross-peak patterns in the spectra. Comparison with in vitro assignments revealed that calbindin D9k (P47M+C80) is initially in the Mg(2+)-bound state, and then gradually converted to the Ca(2+)-bound state. This conversion process initiates after NMR sample preparation. These results showed, for the first time, that cells inside the NMR tube were stressed, presumably because of cell precipitation, the lack of oxygen and nutrients, etc., thereby releasing Ca(2+) into cytosol during the measurements. The results demonstrated that in-cell NMR can monitor the state transitions of stimulated cells through the observation of proteins involved in the intracellular signalling systems. Our method provides a very useful tool for in situ monitoring of the "healthiness" of the cells in various in-cell NMR studies.

Therapeutic strategies for well-differentiated papillary mesothelioma of the peritoneum.

OBJECTIVE: Well-differentiated papillary mesothelioma is an uncommon subtype of mesothelioma with a frequently indolent course, although it occasionally manifests in a more aggressive form. To establish a treatment strategy for this rare disease, we report the clinical characteristics and outcomes of 15 patients with well-differentiated papillary mesothelioma. METHODS: All pathologically diagnosed well-differentiated papillary mesothelioma cases were reviewed between 1998 and 2012. RESULTS: Of the 15 cases, 8 and 7 presented with single and multiple lesions, respectively. All cases with single lesions were asymptomatic, while 4 out of the 7 cases with multiple lesions were symptomatic. After tumor excision, none of the eight single-lesion cases experienced tumor recurrence. Among the other seven cases with multiple lesions, only one patient with disseminated lesions died due to disease burden. Five patients with multiple lesions received cisplatin-based intravenous or intraperitoneal chemotherapy, with a mix of complete (n= 2) and partial (n= 2) responses observed. Of particular note, one patient receiving cisplatin and pemetrexed combination chemotherapy experienced complete tumor resolution without any serious toxicity. CONCLUSIONS: We recommend different treatment strategies based on the disease status. If the tumor is completely resectable, an excisional biopsy seems to be sufficient. If complete resection is unavailable for the asymptomatic patient with a localized tumor extent, close follow-up is an appropriate option. When the tumor is extensive or accompanied by symptoms, chemotherapy should be strongly considered.

Cardiac myxoma with glandular differentiation: an immunohistochemical and ultrastructural study.

A case of cardiac myxoma with glandular differentiation is reported. The patient did not have elements of the Carney triad or syndrome. The tumor was mainly composed of characteristic stellate cells in a focally collagenized, myxoid stroma, along with aggregates of glandular-forming epithelial cells, with mucin-containing intra- and intercellular lumina. Ultrastructurally, these gland spaces displayed short, straight microvilli and junctional complexes. The epithelial cells were positive for cytokeratin 7 and negative for cytokeratin 20. Calretinin was positive in the stellate cells and negative in the epithelial component. The potential origin from pluripotent mesenchymal cells or from seeded stem cells is hypothesized for glandular differentiation in myxomas. Further studies are required to unravel the relationship between stellate cells and the diverse heterologous components reported in these tumors.

Comparative features of calretinin, calbindin, and parvalbumin expressing interneurons in mouse and monkey primary visual and frontal cortices.

Fundamental differences in excitatory pyramidal cells across cortical areas and species highlight the implausibility of extrapolation from mouse to primate neurons and cortical networks. Far less is known about comparative regional and species-specific features of neurochemically distinct cortical inhibitory interneurons. Here, we quantified the density, laminar distribution, and somatodendritic morphology of inhibitory interneurons expressing one or more of the calcium-binding proteins (CaBPs) (calretinin [CR], calbindin [CB], and/or parvalbumin [PV]) in mouse (Mus musculus) versus rhesus monkey (Macaca mulatta) in two functionally and cytoarchitectonically distinct regions-the primary visual and frontal cortical areas-using immunofluorescent multilabeling, stereological counting, and 3D reconstructions. There were significantly higher densities of CB+ and PV+ neurons in visual compared to frontal areas in both species. The main species difference was the significantly greater density and proportion of CR+ interneurons and lower extent of CaBP coexpression in monkey compared to mouse cortices. Cluster analyses revealed that the somatodendritic morphology of layer 2-3 inhibitory interneurons is more dependent on CaBP expression than on species and area. Only modest effects of species were observed for CB+ and PV+ interneuron morphologies, while CR+ neurons showed no difference. By contrast to pyramidal cells that show highly distinctive area- and species-specific features, here we found more subtle differences in the distribution and features of interneurons across areas and species. These data yield insight into how nuanced differences in the population organization and properties of neurons may underlie specializations in cortical regions to confer species- and area-specific functional capacities.

Proteins linked to spatial memory formation of CD1 mice in the multiple T-maze.

In own previous work CD1 mice were tested in the Multiple T-maze (MTM), a robust land maze allowing determination of latency to reach the goal box with food reward and to evaluate correct decisions made on the way to the goal box. Herein, hippocampi of these animals were used for the current study with the aim to investigate differences in protein levels between trained and yoked mice and, moreover, to determine differences in protein levels between trained and yoked mice with and without memory formation in the MTM. Three training sessions were carried out for four training days each, followed by probe trials on Days 5 and 12. Good and no-performers in the MTM were separated based on means and median of latency to reach the goal box on probe trial Day 12. Six hours following the probe trial on Day 12, animals were sacrificed and hippocampi were taken. Proteins were extracted and run on two-dimensional gel electrophoresis, spots were quantified and differentially expressed proteins were identified by mass spectrometry using an ion trap. Levels of 17 proteins were significantly different in trained vs. yoked mice. Seven proteins were differentially expressed comparing trained vs. yoked mice from good and no-performers. A series of proteins were significantly correlated with latency and may link these proteins to spatial memory formation. Differential protein expression in trained vs. yoked mice and in good and no-performers may allow insight into spatial memory formation as well as represent tentative pharmacological targets.

Mitochondrial loss, dysfunction and altered dynamics in Huntington's disease.

Although a direct causative pathway from the gene mutation to the selective neostriatal neurodegeneration remains unclear in Huntington's disease (HD), one putative pathological mechanism reported to play a prominent role in the pathogenesis of this neurological disorder is mitochondrial dysfunction. We examined mitochondria in preferentially vulnerable striatal calbindin-positive neurons in moderate-to-severe grade HD patients, using antisera against mitochondrial markers of COX2, SOD2 and cytochrome c. Combined calbindin and mitochondrial marker immunofluorescence showed a significant and progressive grade-dependent reduction in the number of mitochondria in spiny striatal neurons, with marked alteration in size. Consistent with mitochondrial loss, there was a reduction in COX2 protein levels using western analysis that corresponded with disease severity. In addition, both mitochondrial transcription factor A, a regulator of mtDNA, and peroxisome proliferator-activated receptor-co-activator gamma-1 alpha, a key transcriptional regulator of energy metabolism and mitochondrial biogenesis, were also significantly reduced with increasing disease severity. Abnormalities in mitochondrial dynamics were observed, showing a significant increase in the fission protein Drp1 and a reduction in the expression of the fusion protein mitofusin 1. Lastly, mitochondrial PCR array profiling in HD caudate nucleus specimens showed increased mRNA expression of proteins involved in mitochondrial localization, membrane translocation and polarization and transport that paralleled mitochondrial derangement. These findings reveal that there are both mitochondrial loss and altered mitochondrial morphogenesis with increased mitochondrial fission and reduced fusion in HD. These findings provide further evidence that mitochondrial dysfunction plays a critical role in the pathogenesis of HD.

Chronic electrical stimulation of transected peripheral nerves preserves anatomy and function in the primary somatosensory cortex.

The structure and function of the central nervous system strongly depend on the organization and efficacy of the incoming sensory input. A disruption of somesthetic input severely alters the metabolic activity, electrophysiological properties and even gross anatomical features of the primary somatosensory cortex. Here we examined, in the rat somatosensory cortex, the neuroprotective and therapeutic effects of artificial sensory stimulation after irreversible unilateral transection of a peripheral sensory nerve (the infraorbital branch of the trigeminal nerve). The proximal stump of the nerve was inserted into a silicon tube with stimulating electrodes, through which continuous electrical stimulation was applied for 12 h/day (square pulses of 100 µs, 3.0 V, at 20 Hz) for 4 weeks. Deafferented animals showed significant decreases in cortical evoked potentials, cytochrome oxidase staining intensity (layers II-IV), cortical volume (layer IV) and number of parvalbumin-expressing (layers II-IV) and calbindin-D28k-expressing (layers II/III) interneurons. These deafferentation-dependent effects were largely absent in the nerve-stimulated animals. Together, these results provide evidence that chronic electrical stimulation has a neuroprotective and preservative effect on the sensory cortex, and raise the possibility that, by controlling the physical parameters of an artificial sensory input to a sectioned peripheral nerve, chronically deafferented brain regions could be maintained at near-'normal' conditions. Our findings could be important for the design of sensory neuroprostheses and for therapeutic purposes in brain lesions or neural degenerative processes.

Selective survival of calretinin- and vasoactive-intestinal-peptide-containing nerve elements in human chagasic submucosa and mucosa.

Chronic Chagas' disease is frequently characterized by massive myenteric neuron loss resulting in megacolon with severely and irreversibly disturbed motility. Here, we focused on two submucosal neuron populations, immunoreactive for calretinin (CALR) or somatostatin (SOM), and their respective mucosal nerve fibres in chagasic megacolon. Surgically removed megacolonic segments of seven chagasic patients were compared with seven age- and region-matched non-chagasic control segments. Evaluation included immunohistochemical triple-staining of cryosections for CALR, SOM and peripherin or for CALR and vasoactive intestinal peptide (VIP) and of submucosal whole-mounts for CALR, SOM and the pan-neuronal marker anti-HuC/D. Submucosal neuron counts in chagasic tissue revealed neuron numbers reduced to 51.2 % of control values. In cryosections, nerve fibre area measurements revealed 8.6 % nerve fibre per mucosal area in control segments, but this value decreased to 1.5 % in megacolonic segments. In both evaluations, a disproportionate decrease of SOM-reactive nerve elements was observed. The proportions of SOM-positive neurons related to the total neuron number declined to 2 % (control 10 %) and the proportion of SOM-reactive mucosal nerve fibres related to the whole mucosal area to 0.014 % (control 1.8 %)in chagasic tissue. The second set of cryosections revealed extensive colocalization of CALR with VIP in both surviving submucosal perikarya and mucosal nerve fibres. We suggest that VIP, a neuroprotective and neuroeffectory peptide typically contained in submucosal neurons, allows both the VIP-containing neurons to endure and the patients to survive by maintaining their mucosal barrier, despite the almost complete loss of colonic motility for decades.

Immunohistochemical study of calretinin in normal skin and cutaneous adnexal proliferations.

Calretinin is a calcium-binding protein member of the EF-hand family. The presence of calretinin has been demonstrated in certain stages of the cellular cycle in a wide variety of normal and neoplastic tissues. The main aims of our study were (1) to investigate what structures of the normal skin and cutaneous adnexal proliferations express immunoreactivity for calretinin and (2) to determine the value of immunohistochemical expression for calretinin as a marker for follicular, sebaceous, apocrine, and eccrine differentiation in cutaneous adnexal proliferations. We studied 139 biopsy specimens, including 10 cases of normal skin of different locations and 129 benign and malignant cutaneous adnexal proliferations. In normal skin, we found that calretinin is expressed in the innermost cell layer of the outer root sheath in anagen hair follicle, in both the duct and sebolemma of the sebaceous gland, in the secretory portion of eccrine glands, and in mast cells of the stroma. In cutaneous adnexal proliferations, we found strong immunoreactivity for calretinin in tricholemmal cysts, tricholemmomas/inverted follicular keratoses, tumors of follicular infundibulum, and in some basal cell carcinomas. Focal positivity was also seen in trichoadenomas, trichoblastomas/trichoepitheliomas, pilomatricomas, proliferating tricholemmal tumors, pilar sheath acanthomas, trichofolliculomas, follicular hybrid cysts, cutaneous mixed tumors, steatocystomas, sebaceous hyperplasias, and sebaceomas. These results demonstrate that immunohistochemical study for calretinin may be helpful to identify the innermost cell layer of the outer root sheath in anagen hair follicle and the cutaneous adnexal proliferations showing differentiation toward this structure. Calretinin immunoreactivity supports eccrine differentiation in some sweat gland neoplasms, and it is also useful in identifying neoplasms with ductal sebaceous differentiation.

The diagnostic utility of D2-40, calretinin, CK5/6, desmin and MOC-31 in the differentiation of mesothelioma from adenocarcinoma in pleural effusion cytology.

OBJECTIVE: To evaluate the utility of the lymphatic endothelial marker D2-40, along with calretinin, CK5/6, desmin and MOC-31, in differentiating mesothelioma and adenocarcinoma in pleural effusion cytology. STUDY DESIGN: Forty-five pleural effusion cases representing confirmed reactive effusions (13), mesotheliomas (11) and metastatic adenocarcinomas (21) were immunostained with antibodies against D2-40, calretinin, CK5/6, desmin and MOC-31. RESULTS: D2-40 showed membranous staining in 82% of mesotheliomas and 77% of reactive effusions. Calretinin and CK5/6 were positive in 100 and 64% of mesotheliomas, and 92 and 31% of reactive effusions, respectively. All adenocarcinomas showed lack of staining with these markers. Desmin was negative in all malignant cases and positive in 85% of reactive effusions. All adenocarcinomas were positive for MOC-31 and negative for the remaining markers. CONCLUSION: Calretinin was the most sensitive in detecting mesothelial differentiation, followed by D2-40. Although useful, D2-40 necessitated cautious interpretation due to occasional focal/weak positivity, particularly in limited cellularity samples. The muscle marker desmin was useful in differentiating benign from malignant effusions but not in distinguishing mesotheliomas from adenocarcinomas. MOC-31 was both highly sensitive and specific for detecting adenocarcinoma and was useful as part of a panel of stains in differentiating cells of mesothelial origin from adenocarcinoma.

Calcium-binding proteins typify the dopaminergic neuronal subtypes in the ventral tegmental area of zebra finch, Taeniopygia guttata.

Calcium-binding proteins (CBPs) regulate neuronal function in midbrain dopamine (DA)-ergic neurons in mammals by buffering and sensing the intracellular Ca2+ , and vesicular release. In birds, the equivalent set of neurons are important in song learning, directed singing, courtship, and energy balance, yet the status of CBPs in these neurons is unknown. Herein, for the first time, we probe the nature of CBPs, namely, Calbindin-, Calretinin-, Parvalbumin-, and Secretagogin-expressing DA neurons in the ventral tegmental area (VTA) and substantia nigra (SN) in the midbrain of zebra finch, Taeniopygia guttata. qRT-PCR analysis of ventral midbrain tissue fragment revealed higher Calbindin- and Calretinin-mRNA levels compared to Parvalbumin and Secretagogin. Application of immunofluorescence showed CBP-immunoreactive (-i) neurons in VTA (anterior [VTAa], mid [VTAm], caudal [VTAc]), SN (compacta [SNc], and reticulata [SNr]). Compared to VTAa, higher Calbindin- and Parvalbumin-immunoreactivity (-ir), and lower Calretinin-ir were observed in VTAm and VTAc. Secretagogin-ir was highly localized to VTAa. In SN, Calbindin- and Calretinin-ir were higher in SNc, SNr was Parvalbumin enriched, and Secretagogin-ir was not detected. Weak, moderate, and intense tyrosine hydroxylase (TH)-i VTA neurons were demarcated as subtypes 1, 2, and 3, respectively. While subtype 1 TH-i neurons were neither Calbindin- nor Calretinin-i, ∼80 and ∼65% subtype 2 and ∼30 and ∼45% subtype 3 TH-i neurons co-expressed Calbindin and Calretinin, respectively. All TH-i neuronal subtypes co-expressed Parvalbumin with reciprocal relationship with TH-ir. We suggest that the CBPs may determine VTA DA neuronal heterogeneity and differentially regulate their activity in T. guttata.

Tubal origin of 'ovarian' low-grade serous carcinoma.

Ovarian low-grade serous carcinomas are thought to evolve in a stepwise fashion from ovarian epithelial inclusions, cystadenomas, and borderline tumors. The current study was designed to gain insight into the origins of low-grade serous carcinomas (tubal versus ovarian) by comparatively evaluating the morphologic (secretory and ciliated cell distribution) and immunophenotypic (using antibodies to PAX8, tubulin, calretinin, and Ki67) attributes of its putative precursor lesions, the normal tubal epithelium, and the overt malignancy. A total of 226 adnexal tissues from 178 patients were studied, including 98 adnexae removed for non-neoplastic indications, 48 serous cystadenomas, 42 serous borderline tumors, and 38 low-grade serous carcinomas. Normal distal tubal epithelium comprised an admixture of PAX8+/tubulin- secretory cells and PAX8-/tubulin+ ciliated cells with a proliferative index of ∼3%. The vast majority of ovarian surface epithelia displayed a mesothelial phenotype (calretinin+/PAX8-/tubulin-) and low proliferative index (0% (12 per 1000)), although 4% of cases also displayed foci with tubal phenotype (calretinin-/PAX8+/tubulin+). In contrast, most (78%) of the ovarian epithelial inclusions displayed a tubal phenotype and had a significantly higher proliferative index (1%) than ovarian surface epithelium, indicating that in most cases, the ovarian surface epithelium and ovarian epithelial inclusions are of different lineages. There was a progressive decrease in the population of ciliated cells, as evidenced by increasing secretory/ciliated cell ratio, from ovarian epithelial inclusions/cystadenomas to borderline tumors to low-grade serous carcinoma, indicating that the latter is a clonal expansion of secretory cells. Overall, the findings make a strong argument that the ovarian epithelial inclusions with a tubal phenotype is likely derived from fallopian tube through an intraovarian endosalpingiosis rather than through Mullerian metaplasia from ovarian surface epithelium. Genetic and molecular studies are needed to further confirm this finding as tubal origination of ovarian serous cancers will have a significant impact on ovarian cancer prevention and management.

Calbindin immunoreactivity in the enteric nervous system of larval and adult zebrafish (Danio rerio).

Calbindin is a calcium-binding protein, commonly found in certain subpopulations of the enteric nervous system in mammals. Recently, calbindin-immunoreactive enteric neurons have also been demonstrated in shorthorn sculpin (Myoxocephalus scorpius). In the present study, calbindin immunoreactivity has been investigated in the gut of adult and larval zebrafish (Danio rerio) and differences and similarities between the two species are discussed. Calbindin immunoreactivity is present in 40%-50% of all enteric neurons in adult zebrafish. It first appears at 3 days post-fertilisation (dpf) and is present in all regions of the gut by 13 dpf. Calbindin-immunoreactive nerve cell bodies do not differ in size from calbindin-negative cells. Zebrafish calbindin-immunoreactive neurons are serotonin-negative, with at least some being choline acetyltransferase (ChAT)-positive, in contrast to the sculpin in which cells are generally smaller than the average enteric neuron and are serotonin-positive and ChAT-negative. These findings further emphasise the importance of comparative studies for understanding the diversity of chemical coding in the enteric nervous system of fish and other vertebrates. Improved knowledge of the role of the enteric nervous system is also essential for future studies of gut activity with regard to zebrafish being used as a model organism.

Intravitreal homocysteine-thiolactone injection leads to the degeneration of multiple retinal cells, including photoreceptors.

PURPOSE: Hyperhomocysteinemia is known to cause degeneration of retinal ganglion cells, but its influence on photoreceptors remains largely unknown. In particular, the role of homocysteine-thiolactone (Hcy-T)--the physiologic metabolite of homocysteine that has been proven to be more cytotoxic than homocysteine itself--as a factor that causes retinopathy, has not been defined. This study aimed to investigate the toxic effects of excessive Hcy-T in a mouse model. METHODS: A total of 60 six-week-old female ICR mice were used in this study. The mice were divided into 3 experimental groups and 2 control groups. The mice in the experimental groups were subjected to intravitreal injections of Hcy-T to reach final estimated intravitreal concentrations at 5, 25, and 200 µM, respectively. Mice without injection (blank) and with 0.9 NaCl injections (sham injection) were used as controls. The mice with 200 µM Hcy-T were sacrificed at days 7, 15, 45, and 90 after injection and the mice with 5 or 25 µM Hcy-T were sacrificed at day 90, with the controls sacrificed at day 15 or 90 for comparison. Semi-quantitative dot-blot analysis was performed for confirmation of retinal homocysteinylation. The mouse retinas were evaluated microscopically, with the thickness of total and specific retinal layers determined. Immunohistochemical analysis was performed and the labeled cells were quantified to determine the effects of excessive Hcy-T on specific retinal cells. RESULTS: Dose-dependent retinal homocysteinylation after Hcy-T injection was confirmed. The homocysteinylation was localized in the outer and inner segments of photoreceptors and the ganglion cell layer (GCL). Retinal cell degenerations were found in the GCL, inner nuclear layer, and outer nuclear layer at day 90 after 200 µM Hcy-T injection. Significant thickness reduction was found in the total retina, outer nuclear layer, and the outer and inner segment layers. A trend of thickness reduction was also found in the GCL and inner nuclear layer, although this was not statistically significant. The rhodopsin⁺ photoreceptors and the calbindin⁺ horizontal cells were significantly reduced at day 15, and were nearly ablated at day 90 after 200 µM Hcy-T injection (p<0.001 for both day 15 and day 90), which was not seen in the sham injection controls. The Chx-10⁺ or the Islet-1⁺ bipolar cells and the Pax-6⁺ amacrine cells were severely misarranged at day 90, but no significant reduction was found for both cell types. The GFAP⁺ Müller cells were activated at day 15, but were not significantly increased at day 90 after the injection. CONCLUSIONS: Excessive retinal homocysteinylation by Hcy-T, a condition of hyperhomocysteinemia, could lead to degeneration of photoreceptors, which might lead to retinopathies associated with severe hyperhomocysteinemia or diabetes mellitus.

Expression of calretinin by breast carcinoma and the potential for misdiagnosis of mesothelioma.

AIMS: Calretinin and cytokeratin (CK)5/6 are frequently used to differentiate between metastatic breast cancer and primary malignant mesothelioma in pleural biopsies, but both tumours may express these markers. This study was aimed at evaluating the frequency of calretinin expression in primary breast carcinomas, and assessing the characteristics of the calretinin-positive tumours. METHODS AND RESULTS: Fifty-three primary breast adenocarcinomas were analysed for immunohistochemical expression of calretinin. CK5/6 and epidermal growth factor receptor (EGFR) immunostaining were performed on the calretinin-positive subset. Tumours were classified as basal-like if they met standard morphological and immunohistochemical criteria. Fifteen per cent (8/53) of the breast tumours were positive for calretinin. Eighty-eight per cent (7/8) of the calretinin-positive tumours were grade 3, as compared with 20% (9/45) of the calretinin-negative tumours (P<0.001). Only 13% (1/8) of the calretinin-positive tumours were also oestrogen receptor (ER)-positive, as compared with 87% (39/45) of the calretinin-negative tumours (P<0.001). Eleven per cent (6/53) of the tumours were classified as basal-like. Of these, four were positive for calretinin and two were negative (P=0.003). CONCLUSIONS: Fifteen per cent of breast carcinomas stain with calretinin. These tumours are more likely to be high-grade, ER-negative, and display a basal-like phenotype. These tumours may be misdiagnosed as malignant mesothelioma when they metastasize to the pleura.

Mucinous cystadenoma coexisting with adult granulosa cell tumor in the ovary: is it a composite tumor or heterologous mucinous elements in a granulosa cell tumor?

We herein report a rare tumor combination of mucinous cystadenoma and an adult granulosa cell tumor in the same ovary of a 50-year-old woman. Ultrasound examination showed a multicystic mass in the left ovary. Histologically it showed 2 components that were intimately admixed. One was composed of cysts lined by mucinous epithelium of the intestinal type that was strongly positive for cytokeratin-20 and cytokeratin-7. The other was a granulosa cell tumor of the adult type in which the tumor cells showed microfollicular, sheets, and trabecular patterns. They were positive for calretinin, α-inhibin, and CD99. There are many theories for the histogenesis of such a combination. After discussing all the possibilities we conclude that the mucinous component is probably a heterologous mucinous differentiation within an adult granulosa cell tumor because of the intimate admixture of the 2 components and the mucinous epithelium being of the intestinal type.

Immunohistochemical identification of kidney nephron segments in the dog, rat, mouse, and cynomolgus monkey.

Kidney is a major target organ in preclinical studies. In recent years, intense research has been undertaken to characterize novel renal toxicity biomarkers. In this context, we studied nephron segment specific antibodies against aquaporin-1 (AQP-1), α-glutathione-S-transferase (alpha-GST), Tamm-Horsfall protein (TH), calbindin-D(28K) (CalD), and aquaporin-2 (AQP-2), using an immunoperoxidase method on formalin-fixed paraffin-embedded kidney tissues of dogs, rats, mice, and Cynomolgus monkeys. AQP-1 was specific for proximal tubules and thin descending limbs of Henle's loops and AQP-2 for connecting and collecting ducts in dogs, rats, mice, and Cynomolgus monkeys. Alpha-GST stained the straight part of proximal tubules in dogs and proximal convoluted tubule and straight part of proximal tubules in rats. TH was specific for thick ascending limbs of Henle's loops in mice, rats, dogs, and Cynomolgus monkeys and stained additionally scattered cells in cortical connecting/collecting ducts of dogs. CalD was found in distal convoluted tubules and cortical connecting and collecting ducts of dogs, rats, and mice and in distal convoluted tubules, connecting ducts, and cortical and medullary collecting ducts of Cynomolgus monkey. This panel of antibodies may be a helpful tool to identify renal tubules by light microscopy in preclinical studies and to validate new biomarkers of renal toxicity.

Neurogenesis in the adult human hippocampus.

The genesis of new cells, including neurons, in the adult human brain has not yet been demonstrated. This study was undertaken to investigate whether neurogenesis occurs in the adult human brain, in regions previously identified as neurogenic in adult rodents and monkeys. Human brain tissue was obtained postmortem from patients who had been treated with the thymidine analog, bromodeoxyuridine (BrdU), that labels DNA during the S phase. Using immunofluorescent labeling for BrdU and for one of the neuronal markers, NeuN, calbindin or neuron specific enolase (NSE), we demonstrate that new neurons, as defined by these markers, are generated from dividing progenitor cells in the dentate gyrus of adult humans. Our results further indicate that the human hippocampus retains its ability to generate neurons throughout life.

Exclusion of all three calbindins from a calcium-ferry role in rat enamel cells.

It is widely accepted that healthy enamel formation depends on a steady supply of calcium, yet only fragmentary understanding exists about the mechanisms underlying transepithelial calcium transport. Several lines of evidence indicate that calcium principally follows a transcellular route, which classically is thought to be facilitated by cytosolic calcium-binding proteins termed calbindins. In enamel cells, however, this 'calcium-ferry' dogma appears to fail as we previously found that the major calbindin in murine enamel cells (calbindin-28 kDa) was down-regulated during the peak period of calcium transport and enamel was formed normally in mice lacking calbindin-28 kDa. It remains to be clarified whether the two other known calbindins could function as calcium ferries instead. This study used biochemical and proteomic approaches to obtain definitive identification and quantification of the 30-kDa calbindin (calretinin) and calbindin-9 kDa (S100-G) in enamel epithelium from rat. By establishing that both of these calbindins contribute insufficient calcium capacities in molars and incisors, our results render the calcium-ferry dogma untenable. Of significance to enamel defects and dental bioengineering, these findings support other evidence for an alternative organelle-based mode of calcium transport (calcium transcytosis) and also implicate S100-G/calbindin-9 kDa, but not calretinin, in a calcium-signaling role during enamel maturation.