Cytosolic Calcium-binding Protein Research Articles

#4394TACROLIMUS BUT NOT VOCLOSPORIN INHIBITS KIDNEY TUBULAR CALCIUM AND MAGNESIUM REABSORPTION IN RATS AT CLINICALLY THERAPEUTIC DOSES

Abstract Background and Aims Clinically, the calcineurin inhibitor tacrolimus frequently causes hypercalciuria and hypomagnesemia by inhibiting kidney tubular calcium and magnesium reabsorption. Voclosporin, a novel calcineurin inhibitor approved in the USA and Europe for the treatment of adults with active lupus nephritis, has fewer side-effects including less hypomagnesemia. However, the differences between the kidney tubular effects of tacrolimus and voclosporin are unknown. To address this, we compared the effects of tacrolimus, voclosporin, and vehicle in rats. Method Tacrolimus (0.5 mg/kg) and voclosporin (0.5 mg/kg) were administrated by daily intraperitoneal injections in male Wistar rats and were compared against vehicle (Cremophor EL: 95% ethanol:saline (5:5:90, v:v:v)) for 28 days (n = 8–9/group). Dosages were determined based on pharmacokinetic studies in rats and aimed to achieve the area under the concentration-time curve observed in clinical studies. At day 18, blood and 24 h urine were collected to measure trough levels of the drugs and to analyze the fractional excretions of calcium and magnesium. At sacrifice, kidneys were harvested for immunoblotting of tubular proteins. Results Both tacrolimus and voclosporin reached clinically therapeutic doses with trough levels of 2.4 μg/L ± 0.6 μg/L and 25.8 μg/L ± 9.6 μg/L, respectively. Compared to vehicle, tacrolimus caused significantly higher fractional excretions of calcium (+348% ± 127%, P < 0.001) and magnesium (+60% ± 38%, P < 0.01) and also caused hypomagnesemia (plasma magnesium 0.65 mmol/L ± 0.04 mmol/L vs. 0.81 mmol/L ± 0.02 mmol/L, P < 0.001). Compared to vehicle, voclosporin only caused a slight, but non-statistically significant, increase in fractional calcium excretion (+ 44% ± 38%, P = 0.08) and did not cause higher fractional magnesium excretion (+5% ± 30%, P = 0.96) or hypomagnesemia (plasma magnesium 0.8 mmol/L ± 0.04 mmol/L, P = 0.9). Compared to vehicle, tacrolimus caused an 11-fold decrease in the protein abundance of the cytosolic calcium-binding protein calbindin-D28K and a 2-fold decrease in the abundance of the sodium-chloride cotransporter (NCC). In contrast, voclosporin did not decrease the protein abundances of calbindin-D28K and NCC. No differences were observed between vehicle, tacrolimus, and voclosporin in the calcium channel TRPV5 and magnesium channel TRPM6 (Figure 1). Conclusion In contrast to the calcineurin inhibitor tacrolimus, voclosporin does not inhibit the kidney tubular reabsorption of calcium and magnesium and therefore does not cause hypercalciuria or hypomagnesemia. A possible explanation for this difference is that tacrolimus but not voclosporin affects tubular transport in the distal convoluted tubule, which was further supported by the selective inhibition of calbindin-D28K and NCC by tacrolimus. Our data show that tubulotoxicity of tacrolimus is not apparent with voclosporin treatment, at clinically relevant doses.

Potential Neuroprotective Role of Calretinin-N18 and Calbindin-D28k in the Retina of Adult Zebrafish Exposed to Different Wavelength Lights.

The incidence rates of light-induced retinopathies have increased significantly in the last decades because of continuous exposure to light from different electronic devices. Recent studies showed that exposure to blue light had been related to the pathogenesis of light-induced retinopathies. However, the pathophysiological mechanisms underlying changes induced by light exposure are not fully known yet. In the present study, the effects of exposure to light at different wavelengths with emission peaks in the blue light range (400-500 nm) on the localization of Calretinin-N18 (CaR-N18) and Calbindin-D28K (CaB-D28K) in adult zebrafish retina are studied using double immunofluorescence with confocal laser microscopy. CaB-D28K and CaR-N18 are two homologous cytosolic calcium-binding proteins (CaBPs) implicated in essential process regulation in central and peripheral nervous systems. CaB-D28K and CaR-N18 distributions are investigated to elucidate their potential role in maintaining retinal homeostasis under distinct light conditions and darkness. The results showed that light influences CaB-D28K and CaR-N18 distribution in the retina of adult zebrafish, suggesting that these CaBPs could be involved in the pathophysiology of retinal damage induced by the short-wavelength visible light spectrum.

Parvalbumin immunohistochemical expression in the spectrum of perivascular epithelioid cell (PEC) lesions of the kidney.

Parvalbumin is a cytosolic calcium-binding protein expressed in the distal convoluted tubule of the renal nephron. Among epithelial renal tumors, the reactivity for parvalbumin is observed in chromophobe renal cell carcinomas and frequently in oncocytomas. On the other hand, there are no data available on parvalbumin expression in the mesenchymal tumors of the kidney. Therefore, the purpose of this study was to evaluate the expression of parvalbumin in the spectrum of PEC (perivascular epithelioid cells) lesions of the kidney. Sixty-six PEC lesions (37 classic angiomyolipomas, 10 microscopic angiomyolipomas, 7 epithelioid angiomyolipomas/pure epithelioid PEComas, 5 leiomyoma-like angiomyolipomas, 3 lipoma-like angiomyolipomas, 2 intraglomerular lesions, 1 angiomyolipoma with epithelial cysts (AMLEC), and 1 sclerosing angiomyolipoma) were immunohistochemically stained with parvalbumin. Overall, parvalbumin immunostain was found in fifty-six PEC lesions (85%) and absent in the remaining ten cases (15%). Classic angiomyolipomas were positive in almost all cases (97%). Intraglomerular lesions and AMLEC showed parvalbumin immunolabeling as well. None of the 7 epithelioid angiomyolipomas/pure epithelioid PEComas or the only sclerosing angiomyolipoma expressed parvalbumin. In conclusion, we demonstrated the immunolabeling of parvalbumin in almost all PEC lesions of the kidney, but not in the epithelioid angiomyolipoma/pure epithelioid PEComa. This finding could shed light on some biological characteristics observed in the PEC lesions such as the plasticity of their cellular component. Moreover, parvalbumin may be another useful tool in the differential diagnosis among epithelioid angiomyolipoma/pure epithelioid PEComa with other renal eosinophilic tumors, such as oncocytoma and chromophobe renal cell carcinoma.

The Role of S100B Protein at 24 Hours of Postnatal Age as Early Indicator of Brain Damage and Prognostic Parameter of Perinatal Asphyxia.

Introduction. S100B protein is a cytosolic calcium-binding protein with a molecular weight of 21 kDa, which is present in various cells and concentrated mainly in the glial cells, which play a vital role in the maintenance of cellular homeostasis in the central nervous system. It is possible that increased S100B protein level might be considered as sensitive and specific indicator to predict early brain damage. Aim. To investigate the prognostic value of serum S100B protein in neonates with perinatal asphyxia (PA) at 24 hours of postnatal age. Methods. A systematic review was performed. Inclusion criteria were studies including data of neonates with PA, monitored with serum S100B, and with neurodevelopmental follow-up of at least 2 weeks. The period of bibliographic search was until January 2017. The consulted databases were MEDLINE, PsycINFO, and Embase. A combination of the following subject headings and keywords was adapted for each electronic database: “perinatal asphyxia,” “hypoxic ischemic encephalopathy,” “hypoxia-ischemia, brain,” and “S100B.” Meta-Disc1.4 software was used. Results. From the 1620 articles initially identified, 6 were finally included and reviewed. The overall diagnostic sensitivity of serum S100B was 0.80 (95% confidence interval [CI] = 0.68-0.88) and the specificity was 0.79 (95% CI = 0.70-0.87). But there was lower predictability value, that is, the positive likelihood ratio was only 3.26 (95% CI 1.74-6.12) and the negative likelihood ratio was 0.32 (95% CI = 0.20-0.5). The diagnostic odds ratio was 12.40 (95% CI = 4.66-33.0). Conclusion. Increased serum S100B level at 24 hours of postnatal life can demonstrate brain damage, but it should not be the only one used to predict PA outcome.

Decreased expression of annexin A10 in gastric cancer and its overexpression in tumor cell growth suppression.

Gastric carcinoma is the most common neoplasm in Southeast Asian populations and is the second leading cause of cancer death worldwide. Annexins are a family of cytosolic calcium and membrane binding proteins that have been implicated in a wide variety of cell functions. Recent studies have suggested that Annexin A10 (ANXA10), a member of the Annexin protein family, is down-regulated in specific types of cancer. However, the underlying molecular mechanisms of the dysregulation of ANXA10 remain to be elucidated. In the present study, to investigate the biological effects of ANXA10 on gastric carcinoma, aberrant expression of ANXA10 was evaluated by Western blot analysis, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC), in gastric cancer tissues and cell lines. Decreased expression of ANXA10 was observed in five selected gastric cancer tissues compared to the normal surrounding mucosa. In the cancer cell lines, seven out of nine selected gastric cancer cell lines had no detectable ANXA10 by RT-PCR. Among these, when an ANXA10 expressing plasmid was introduced into MKN-1 cells, cell growth was suppressed and apoptosis augmented. The results of this study demonstrated that ANXA10 was aberrantly regulated in gastric carcinoma and suggests that down-regulation of ANXA10 might be involved in gastric carcinogenesis. In addition, ANXA10 may play a role, as a tumor suppressor, in the development and progression of gastric cancer.

Role of calcium-binding sites in calcium-dependent membrane association of annexin A4.

Annexin A4 (Anx4) is a cytosolic calcium-binding protein with four repeat domains, each containing one calcium-binding site (CBS). The protein interacts with the phospholipid membrane through the CBS-coordinated calcium ion, although the role of each CBS in the calcium-dependent association is unclear. To determine the role of each CBS, 15 CBS-abolished variants were produced in various combinations by substitution of a calcium-liganding residue on each CBS by Ala. Various mutant combinations produced different influences on calcium-dependent membrane-binding behavior and on the sodium-dependent dissociation of membrane-bound Anx4. Our data suggest the interaction of Anx4 with the lipid membrane consists of strong and weak interactions. CBSs I and IV mediate formation of strong interactions, while CBSs II and III are important for weak interactions. We also suggest Anx4 binds the lipid membrane through CBSs I and IV in the cytoplasmic fluids.

S100B Induces the Release of Pro-Inflammatory Cytokines in Alveolar Type I-like Cells

S100B, a 21kDa cytosolic calcium-binding protein of the EF-hand type, present in high abundance in the brain, stimulates inflammatory responses in different cellular types inside and outside the central nervous system. Most of extracellular S100B effects are mediated by Receptor for Advanced Glycation End-products (RAGE). RAGE is highly expressed in lung by Alveolar Type-I (AT-I) cells and its activation contributes to ALI/ARDS pathogenesis. In this in-vitro study, we tested the hypothesis that S100B stimulates an ATI-derived cell line (R3/1) to secrete inflammatory mediators involved in lung inflammation. Our main result is that S100B stimulates R3/1 cells to secrete TNF-alpha and IL-6 (well-known pro-inflammatory cytokines in lung inflammation and neurogenic pulmonary edema), but not sICAM-1, CINC-1 or CINC-3. Soluble RAGE (sRAGE) reduced S100B-dependent secretion of TNF-alpha but did not decrease S100B-dependent secretion of IL-6. Moreover, in absence of S100B, sRAGE enhanced IL-6 release. This study demonstrates that in vitro S100B dose-dependently stimulated R3/1 cells, to enhance the secretion of TNF-alpha and IL-6; S100B pro-inflammatory activity might be mediated at least in part by RAGE. Besides acting as decoy receptor, sRAGE could have pro-inflammatory properties.

What Determines the Kinetics of the Slow Afterhyperpolarization (sAHP) in Neurons?

What Determines the Kinetics of the Slow Afterhyperpolarization (sAHP) in Neurons?

Immunohistochemical study of the distribution of calcium binding proteins in the brain of a chondrostean (Acipenser baeri)

Chondrosteans represent an ancient lineage in ray-finned bony fishes and hence in jawed vertebrates. This immunohistochemical study in the brain of the Siberian sturgeon reports the neuronal distribution of three cytosolic calcium-binding proteins: calbindin-D28k (CB), calretinin (CR), and parvalbumin (PV). CB and CR are widely expressed in different neuron subsets distributed throughout the sturgeon brain. Studies using double immunofluorescence reveal a wide co-distribution of CB and CR in the brain nuclei but scarce co-localization at cellular level. In the forebrain, CR- and CB-immunoreactive (ir) populations were observed in the olfactory bulbs, in pallial and subpallial telencephalic areas, and in some diencephalic nuclei. CR-ir cells were also observed in the posterior tubercle and CB-ir cells in the preglomerular complex. At midbrain and hindbrain basal levels, CB-ir and CR-ir cell bodies were mainly distributed in periventricular areas. In the cerebellum, CB and CR cells were co-localized in some granular cell subsets in laterodorsal and dorsolateral regions, and in some Purkinje-like cells. CB-ir and CR-ir fibers were mainly observed in the olfactory bulbs, hypothalamus, and habenula, and in fiber tracts that coursed in the optic tectum and through the mesencephalic and rhombencephalic basal areas. With regard to PV, the sturgeon brain showed a rather limited distribution of PV-ir perikarya and fibers. Thus, CR, CB, and PV allowed the identification of subpopulations of neurons not distinguished on the basis of cytoarchitecture alone, which provided a better understanding of the anatomical organization of the sturgeon brain. These results reveal numerous shared features with teleosts, but also important differences.

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.

In vitro studies on intestinal calcium and phosphate transport in horses

Transepithelial transport mechanisms play a key role in regulating the absorption and secretion of calcium (Ca 2 + ) and inorganic phosphate (P i) in the gastrointestinal tract. Although intestinal disorders with imbalances in macromineral homeostasis are frequently observed in horses, available data on intestinal Ca 2 + and P i transport are limited. The aim of the present study was to characterize the intestinal Ca 2 + and P i transport functionally by using the in vitro radioisotope tracer technique with Ussing chambers and to identify components involved in Ca 2 + transport at both mRNA and protein level. Among the different intestinal segments, the duodenum showed significant and highest active Ca 2 + absorption. The findings from RT-PCR and Western blot analysis suggest that the epithelial Ca 2 + channel TRPV6, the cytosolic calcium binding protein calbindin-D 9K and the plasma membrane calcium ATPase PMCA may be involved in active transcellular Ca 2 + transport. Regarding the P i transport, the results indicate significant active P i secretion in the jejunum, but the contributing mechanisms remain unclear. A significant inhibiting effect of ouabain as an antagonist of the basolateral Na +/K +-ATPase on the serosal-to-mucosal P i transport suggests a pivotal role of Na + in jejunal P i transport in the horse.

Protein synthesis of the pro-inflammatory S100A8/A9 complex in plasmacytoid dendritic cells and cell surface S100A8/A9 on leukocyte subpopulations in systemic lupus erythematosus

IntroductionSystemic lupus erythematosus (SLE) is an autoimmune disease with chronic or episodic inflammation in many different organ systems, activation of leukocytes and production of pro-inflammatory cytokines. The heterodimer of the cytosolic calcium-binding proteins S100A8 and S100A9 (S100A8/A9) is secreted by activated polymorphonuclear neutrophils (PMNs) and monocytes and serves as a serum marker for several inflammatory diseases. Furthermore, S100A8 and S100A9 have many pro-inflammatory properties such as binding to Toll-like receptor 4 (TLR4). In this study we investigated if aberrant cell surface S100A8/A9 could be seen in SLE and if plasmacytoid dendritic cells (pDCs) could synthesize S100A8/A9.MethodsFlow cytometry, confocal microscopy and real-time PCR of flow cytometry-sorted cells were used to measure cell surface S100A8/A9, intracellular S100A8/A9 and mRNA levels of S100A8 and S100A9, respectively.ResultsCell surface S100A8/A9 was detected on all leukocyte subpopulations investigated except for T cells. By confocal microscopy, real-time PCR and stimulation assays, we could demonstrate that pDCs, monocytes and PMNs could synthesize S100A8/A9. Furthermore, pDC cell surface S100A8/A9 was higher in patients with active disease as compared to patients with inactive disease. Upon immune complex stimulation, pDCs up-regulated the cell surface S100A8/A9. SLE patients had also increased serum levels of S100A8/A9.ConclusionsPatients with SLE had increased cell surface S100A8/A9, which could be important in amplification and persistence of inflammation. Importantly, pDCs were able to synthesize S100A8/A9 proteins and up-regulate the cell surface expression upon immune complex-stimulation. Thus, S100A8/A9 may be a potent target for treatment of inflammatory diseases such as SLE.

Transcriptional and translational expression of calbindin-D9k in the duodenum, kidney and uterus of a female canine model

Calbindin-D9k (CaBP-9k) is a cytosolic calcium-binding protein expressed in tissues in the intestine, uterus, placenta, kidney, pituitary gland and bone. Its exact function is unknown, but it is considered to regulate intracytoplasmic concentration and transport of free ions (Ca2+). CaBP-9k protein is involved in intestinal calcium absorption in the intestine and in the regulation of myometrial activity by intracellular calcium in the uterus. Renal CaBP-9k protein is expressed at the site of calcium re-absorption in the kidney and expressed in distal convoluted tubules, where it is thought to facilitate calcium re-absorption. Expression of the CaBP-9k gene has been explored in most mammalians except in a canine model. Presently, we elucidated the expression of CaBP-9k mRNA and protein in the duodenum, kidney and uterus in a canine model involving two adult (2.5-year-old) female beagles. To collect tissues, the dogs were euthanized and then the abdominal cavity was exposed by midline incision. The proximal duodenum, cortex of kidney and uterine horn were collected. Expression of CaBP-9k mRNA was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. CaBP-9k protein expression and localization were ascertained by Western blot analysis and immunohistochemistry, respectively. CaBP-9k mRNA was detected in the duodenum, but not in the kidney and uterus. Its protein was expressed only in the enterocytes of the duodenum. Taken together, the results indicate that CaBP-9k mRNA and protein are highly expressed in the enterocytes of the duodenum of a canine model, consistent with findings in other mammalian species.

Calcium Homeostasis and Regulation of Calbindin-D9kby Glucocorticoids and Vitamin D as Bioactive Molecules

Abstract － Calbindin-D 9k (CaBP-9k), a cytosolic calcium-binding protein, is expressed in a variety of tissues, i.e., the duodenum, uterus, placenta, kidney and pituitary gland. Duodenal CaBP-9k is involved in intestinal calcium absorption, and is regulated at transcriptional and post-transcriptional levels by 1,25- dihydroxyvitamin D3, the hormonal form of vitamin D, and glucocorticoids (GCs). Uterine CaBP-9k has been implicated in the regulation of myometrial action(s) through modulation of intracellular calcium, and steroid hormones appear to be the main regulators in its uterine and placental regulation. Because phenotypes of CaBP-9k-null mice appear to be normal, other calcium-transporter genes may compensate for its gene deletion and physiological function in knockout mice. Previous studies indicate that CaBP-9k may be controlled in a tissue-specific fashion. In this review, we summarize the current information on calcium homeostasis related to CaBP-9k gene regulation by GCs, vitamin D and its receptors, and its molecular regulatory mechanism. In addition, we present related data from our current research.Keywords: Calcium-binding proteins, Calbindin-D9k and vitamin D

Induction of calprotectin mRNAs by lipopolysaccharide in the salivary gland of mice

Calprotectin is a major cytosolic calcium-binding protein of leukocytes which belongs to the S100 protein family. S100A8 and S100A9, major types of calprotectin are heterodimeric complexes being composed of light- and heavy-chain subunits. The calprotectin levels in the plasma, feces, synovial fluid, gingival crevicular fluid, dental calculus and saliva change when the host animal suffers from several inflammatory diseases. Members of Toll-like receptor (TLR) family are pattern-recognition receptors for lipopolysaccharide (LPS) and other pathogens. Here we examined if the biological role of TLR receptor is reflected to the calprotectin expression in the salivary gland. Time course study by using real-time RT-PCR detected higher levels of S100A8 and S100A9 mRNA at 1.5-3 h after injection of LPS in both the submandibular gland (SMG) and parotid gland (PG) of C3H/HeN mice but not in the same tissues of C3H/HeJ, a TLR-4 mutant strain, indicating that this induction is mediated via the TLR-4. These results indicate that, an inflammatory marker, calprotectin, is expressed in the mouse salivary gland and that LPS stimulated its synthesis. Calprotectin (S100A8/A9) showed minimum expression in all cellular segments in the SMG except excretory duct cells, which showed strong signal at the cytoplasm. LPS induced their expressions in the granular convoluted tubular cells and striated duct cells. In the PG, these proteins were expressed very weakly in both duct and acinar cells with a little stronger staining for the former cells. LPS injection induced calprotectin (S100A8/A9) in both duct and acinar cells especially in the former cells.

Molecular mechanism of regulation of the calcium‐binding protein calbindin‐D9k,and its physiological role(s) in mammals: a review of current research

Calbindin-D9k (CaBP-9k) is a cytosolic calcium-binding protein that is expressed in a variety of tissues, such as uterus, placenta, intestine, kidney, pituitary gland and bone. At present, the precise role(s) of CaBP-9k remains to be clarified. CaBP-9k-null mice are normal, which indicates that other calcium-transporter genes can compensate for the lack CaBP-9k. Uterine CaBP-9k has been shown to be involved in the regulation of myometrial activity by intracellular calcium. In the uterus and placenta, CaBP-9k expression is regulated by the sex steroid hormones oestrogen (E2) and progesterone (P4). Intestinal CaBP-9k is involved in intestinal calcium absorption, and is regulated at the transcriptional and post-transcriptional levels by 1,25-dihydroxyvitamin D3, the hormonal form of vitamin D. Thus, evidence to date suggests that CaBP-9k may be regulated in a tissue-specific manner. In this review, we will summarize current data on the molecular mechanism of regulation of CaBP-9k in mammals, including recent research data generated in our laboratories.

A Model for Auditory Brain Stem Implants: Bilateral Surgical Deafferentation of the Cochlear Nuclei in the Macaque Monkey

Patients with extensive bilateral lesions of the auditory nerve have a profound and irreversible sensorineural hearing loss (SNHL), which can only be overcome with individually-fitted auditory brain stem implants that directly stimulate the cochlear nuclei. Despite the enormous potential of this increasingly applied treatment, the auditory performance of many implanted patients is limited, and the variability between cases hinders a complete understanding of the role played by the multiple parameters related to the efficacy of the implant. To mimic the condition of patients who have bilateral lesions of the auditory nerve, we developed an experimental model of bilateral deafferentation of the cochlear nuclei by surgical transection of the cochlear nerves of adult primates. We performed bilateral transection of the cochlear nerves of six adult, healthy, male captive-bred macaques (Macaca fascicularis). Before surgery, brain stem auditory evoked potentials were recorded. The histological material obtained from these animals was compared with similarly processed sections from seven macaques with intact cochlear nerves. The surgical technique, similar to that used in human neuro-otology, combined a labyrinthectomy and a neurectomy of the cochlear nerves, and caused deafness. We analyzed immunocytochemically the expression in cochlear nerve fibers of neurofilaments (SMI-32), and cytosolic calcium binding proteins calretinin, parvalbumin and calbindin, and also applied a histochemical reaction for acetylcholinesterase. None of the primates had any major complications due to the surgical procedure. The lesions produced massive anterograde degeneration of the cochlear nerves, evidenced by marked gliosis and by loss of both type I fibers (which in this species are immunoreactive for calretinin, parvalbumin and neurofilaments) and type II fibers (which are acetylcholinesterase positive). The model of surgical transection described herein causes extensive damage to the cochlear nerves while leaving the cochlea intact, thus mimicking the condition of patients with profound SNHL due to bilateral cochlear nerve degeneration. The phylogenetic proximity of primates to humans, and the paramount advantage of close anatomical and physiological similarities, allowed us to use the same surgical technique applied to human patients, and to perform a thorough evaluation of the consequences of neurectomy. Thus, bilateral surgical deafferentation of the macaque cochlear nuclei may constitute an advantageous model for study of auditory brain stem implants.

Modulation of the dynamics of mitochondrial calcium by buffer cytosolic proteins and plasma membrane fluxes

Buffer performance of mitochondria in intracellular calcium signaling is studied in relation to the amount of cytosolic calcium-binding proteins and calcium ion fluxes across the plasma membrane of the cell.

Calbindin-D9k mRNA Expression in the Rat Uterus Following Exposure to Methoxychlor: A Comparison of Oral and Subcutaneous Exposure

Calbindin-D(9k) (CaBP-9k) is a cytosolic calcium-binding protein that is induced by estrogenic compounds possibly through estrogen receptors. We compared CaBP-9k mRNA expression in the uterus with uterotrophic response in immature rats exposed to methoxychlor (MC), an environmental chemical with estrogenic activity. MC was orally or subcutaneously administered to 3-week-old female Sprague-Dawley rats for 3 days. The weights of the uterus and vagina significantly increased in the oral treatment group at a dose of 50, 100 and 200 mg/kg, but those of the subcutaneous (SC) treatment group only increased at 200 mg/kg. Northern blot analysis showed that CaBP-9k mRNA expression was significantly induced in a dose-dependent manner at doses of 50, 100 and 200 mg/kg/day in the oral treatment group. SC administration of MC induced significant expression at only a dose of 200 mg/kg/day; this was similar to the uterotrophic response. MC has an estrogenic effect on the uterus as shown by the increase in weight and induction of CaBP-9k mRNA expression, which were much greater following exposure via oral gavage than via the SC route. The strong correlation between the results of in vivo uterotrophic assay and CaBP-9k mRNA expression suggests that CaBP-9k mRNA expression in the rat uterus may be used as an early gene marker for detection of the estrogenic effects of putative environmental chemicals.

ApoE genotype effects—A challenge for many

A major issue addressed by the Peers’ commentaries (and central in Spires’ and Hyman’s contribution) is related to the influence of the apoE polymorphism on spines. Spines which are highly organized substructures of many dendrites, especially of the principal cortical neurons, i.e. the pyramidal cells, represent a major relay between presynaptic and postsynaptic parts of the neuronal network. Thus, it is not surprising to see a strong inverse correlation between synapse numbers and cognitive decline in Alzheimer’s disease. The role of the apoE-polymorphism in the observed changes of numeric spine densities in both Alzheimer’s disease and animal models thereof, however, is not clear. It remains an open question whether the apoE4-associated reduction in spine numbers is due to a developmental impairment of the dendrites proper or, alternatively, because afferents (i.e. axons) are lacking which normally would require spines as the postsynaptic partner. A reduction of afferents may be consequence of partial deafferentation (during a post-developmental challenge) or when afferents were not formed (during development). Both might transneuronally lead to fewer spines. Our study, however, deals with aspiny neurons and is therefore not suited to address the important topic of apoE’s role in spine formation. The neuronal population we selected to study is rich in Parvalbumin, a cytosolic calcium-binding protein. This might contribute to the similar dendritic phenotype observed when considering the apoE genotype as a variable. Belinson and co-workers point to the fact that experimental studies – which found apoE-isoform related effects on neuritic length and arborisation – showed that the effects are blocked by antagonists of the apoE-receptor LDL-related receptor (LRP) suggesting an effect downstream of ApoE-binding. This raises the interesting question whether Parvalbumin