Inhibition Of Calcium Transport Research Articles

Modulation of fibroblast growth factor-23 expression and transepithelial calcium absorption in Caco-2 monolayer by calcium-sensing receptor and calcineurin under calcium hyperabsorptive state

Fibroblast growth factor (FGF)-23 and calcium-sensing receptor (CaSR) have previously been postulated to be parts of a negative feedback regulation of the intestinal calcium absorption to prevent excessive calcium uptake and its toxicity. However, the underlying mechanism of this feedback regulation remained elusive, especially whether it required transcription of FGF-23. Herein, we induced calcium hyperabsorptive state (CHS) by exposing intestinal epithelium-like Caco-2 monolayer to 30 mM CaCl2 and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] after which FGF-23 mRNA levels and transepithelial calcium flux were determined. We found that CHS upregulated FGF-23 transcription, which was reverted by CaSR inhibitors (Calhex-231 and NPS2143) but without effect on CaSR transcription. Although 10 nM 1,25(OH)2D3 was capable of enhancing transepithelial calcium flux, the higher-than-normal calcium inundation as in CHS led to a decrease in calcium flux, consistent with an increase in FGF-23 protein expression. Administration of inhibitors (≤10 μM CN585 and cyclosporin A) of calcineurin, a mediator of CaSR action to control transcription and production of its target proteins, was found to partially prevent FGF-23 protein production and the negative effect of CHS on calcium transport, while having no effect on FGF-23 mRNA expression. Direct exposure to FGF-23, but not FGF-23 + PD173074 (FGFR1/3 inhibitor), also completely abolished the 1,25(OH)2D3-enhanced calcium transport in Caco-2 monolayer. Nevertheless, CHS and CaSR inhibitors had no effect on the mRNA levels of calcineurin (PPP3CB) or its targets (i.e., NFATc1–4). In conclusion, exposure to CHS induced by high apical calcium and 1,25(OH)2D3 triggered a negative feedback mechanism to prevent further calcium uptake. CaSR and its downstream mediator, calcineurin, possibly contributed to the regulatory process, in part by enhancing FGF-23 production to inhibit calcium transport. Our study, therefore, corroborated the physiological significance of CaSR-autocrine FGF-23 axis as a local feedback loop for prevention of excessive calcium uptake.

Antispasmodics for Chronic Abdominal Pain: Analysis of North American Treatment Options.

Chronic abdominal pain is a common gastrointestinal (GI) symptom that characterizes many functional GI disorders/disorders of gut-brain interaction, including irritable bowel syndrome, functional dyspepsia, and centrally mediated abdominal pain syndrome. The symptoms of abdominal pain in these highly prevalent disorders are often treated with antispasmodic agents. Antispasmodic treatment includes a broad range of therapeutic classes with different mechanisms of action, including anticholinergic/antimuscarinic agents (inhibition of GI smooth muscle contraction), calcium channel inhibitors (inhibition of calcium transport into GI smooth muscle), and direct smooth muscle relaxants (inhibition of sodium and calcium transport). The aim of this review article was to examine the efficacy and safety of antispasmodics available in North America (e.g., alverine, dicyclomine, hyoscine, hyoscyamine, mebeverine, otilonium, pinaverium, and trimebutine) for the treatment of chronic abdominal pain in patients with common disorders of gut-brain interaction. For the agents examined, comparisons of studies are limited by inconsistencies in treatment dosing and duration, patient profiles, and diagnostic criteria employed. Furthermore, variability in study end points limits comparisons. Risk of selection, performance, detection, attrition, and reporting bias also differed among studies, and in many cases, risks were considered “unclear.” The antispasmodics evaluated in this review, which differ in geographic availability, were found to vary dramatically in efficacy and safety. Given these caveats, each agent should be considered on an individual basis, rather than prescribed based on information across the broad class of agents.

HAMLET, a protein complex from human milk has bactericidal activity and enhances the activity of antibiotics against pathogenic Streptococci.

HAMLET is a protein-lipid complex derived from human milk that was first described for its tumoricidal activity. Later studies showed that HAMLET also has direct bactericidal activity against select species of bacteria, with highest activity against Streptococcus pneumoniae Additionally, HAMLET in combination with various antimicrobial agents can make a broader range of antibiotic-resistant bacterial species sensitive to antibiotics. Here, we show that HAMLET has direct antibacterial activity not only against pneumococci, but also against Streptococcus pyogenes (GAS) and Streptococcus agalactiae (GBS). Analogous to pneumococci, HAMLET-treatment of GAS and GBS resulted in depolarization of the bacterial membrane followed by membrane permeabilization and death that could be inhibited by calcium and sodium transport inhibitors. Treatment of clinical antibiotic-resistant isolates of S. pneumoniae, GAS, and GBS with sublethal concentrations of HAMLET in combination with antibiotics decreased the minimal inhibitory concentrations of the respective antibiotic into the sensitive range. This effect could also be blocked by ion transport inhibitors, suggesting that HAMLET's bactericidal and combination treatment effects used similar mechanisms. Finally, we show that HAMLET potentiated the effects of erythromycin against erythromycin-resistant bacteria more effectively than it potentiated killing by penicillin G of bacteria resistant to penicillin G. These results show for the first time that HAMLET effectively kills three different species of pathogenic Streptococci using similar mechanisms and also potentiate the activity of macrolides and lincosamides more effectively than combination treatment with beta-lactams. These findings suggest a potential therapeutic role for HAMLET in repurposing antibiotics currently causing treatment failures in patients.

Ocean acidification: effects of pH on 45Ca uptake by lobster branchiostegites.

Gill chambers of the Atlantic lobster, Homarus americanus, possess three structures that are involved with respiration and ion regulation: gill filaments, epipodites, and branchiostegites. This paper describes ion transport mechanisms present in the plasma membranes of branchiostegite epithelial cells and the effects of pH on the uptake of 45Ca by these processes. Partially purified membrane vesicles (PPMV) of branchiostegite cells were produced by a homogenization/centrifugation method that has previously been used to define ion transport processes in both crab and lobster gill tissues. In the present study, lobster branchiostegite PPMV 45Ca uptake was highest at pH 8.5 and lowest at pH values between 6.0 and 7.0 (p < 0.02). At pH 8.0, 45Ca uptake was a biphasic process consisting of a saturable process at low [Ca] and a linear process at higher [Ca]. At pH 6.0, 45Ca uptake was only a linear process and paralleled linear uptake at pH 8.0. A valinomycin/K+-induced membrane potential (PD, inside negative) doubled 45Ca uptake at pH 7.0 above that in the absence of a PD (p < 0.05). An induced PD at pH 8.0 did not significantly (p > 0.05) affect 45Ca uptake observed in the absence of a PD, but was threefold greater than uptake at pH 7.0 in the absence of a PD (p < 0.05). Amiloride (2mM) did not affect 45Ca uptake at pH 8.0, but 2mM amiloride + 100µM verapamil reduced uptake by approximately 50%. In the presence of both 2mM amiloride + 100µM verapamil, 15s 45Ca influx at pH 8.5 was a hyperbolic function of [Ca] (0.1-5mM) (Km = 4.2 ± 0.3mM; Jmax = 9792 ± 439pmol/mg protein × 15s). 45Ca influxes at pH 7.5 under the same conditions were also hyperbolic with Km = 8.3 ± 1.4mM; Jmax = 10732 ± 1250pmol/mg protein × 15s. Km values were significantly different (p < 0.05), but Jmax values were not (p > 0.05). These results suggest that 45Ca uptake by lobster branchiostegites may have occurred by the combination of diffusion through a verapamil-inhibited calcium channel and carrier-mediated transport by amiloride-insensitive, electroneutral, 1Ca2+/2H+ antiporters. Decreased pH, as might occur during ocean acidification, did not appear to modify calcium diffusion through the channels, but protons acted as competitive inhibitors of calcium transport by carrier-mediated antiport. Decreased calcium uptake with continued ocean acidification may significantly affect calcification processes during periodic molting, potentially influencing mortality.

Effect of Calcium Flux on Filopodia of Epithelial Cells

Filopodia are the sensors of a cell and are responsible for directing cell motility. Like “antennae,” they receive signals from both soluble ligands in the extracellular mileau and those attached to the substrate. In the nerve axon, these two aspects of signaling are integrated so as to modulate chemotactic signaling according to the composition of the substrate. The role of filopodia in the modulation is not clear. Although epithelial cells differ from nerve cells in their organization, the same second messengers mediate the filopodia's sensory function. In both cell types, activation of a PKC (protein kinase C) with a tumor promoter inhibited filopodia or caused their dissolution. Because Ca++ selectively activates one subset of PKCs, local Ca++ elevation could activate these isozymes and also thereby decrease filopodia. Previous reports showed contradictory results for Ca++ second messenger, however, in the axon, i.e. positive or negative effects on filopodia. Here, we investigate how filopodia dynamics are affected by calcium. We measured percentage of the cell periphery covered with filopodia and percentage of cells showing filopodia. There is an inhibitor of filopodia in culture media, so that replacing the medium with a buffer increased filopodia. This occurred regardless of whether calcium was present in the buffer or absent. When cells remained in the medium, however, inhibitors of calcium transport caused a decrease in filopodia. Cyclopiazonic acid in Ca++-free buffer typically blocks uptake of Ca++ into the endoplasmic reticulum (ER) and causes net Ca++ efflux. This enhanced filopodia. Restoring extracellular Ca++ in the extracellular buffer after depleting the ER store also enhanced filopodia production. We conclude that filopodia display was sensitive to calcium flux but not to absolute physiologically relevant calcium concentrations. It remains to be determined how calcium flux is detected in epithelial cells.

Structure and Function of Caltrin (Calcium Transport Inhibitor) Proteins.

Caltrin (calcium transport inhibitor) is a family of small and basic proteins of the mammalian seminal plasma which bind to sperm cells during ejaculation and inhibit the extracellular Ca2+ uptake, preventing the premature acrosomal exocytosis and hyperactivation when sperm cells ascend through the female reproductive tract. The binding of caltrin proteins to specific areas of the sperm surface suggests the existence of caltrin receptors, or precise protein-phospholipid arrangements in the sperm membrane, distributed in the regions where Ca2+ influx may take place. However, the molecular mechanisms of recognition and interaction between caltrin and spermatozoa have not been elucidated. Therefore, the aim of this article is to describe in depth the known structural features and functional properties of caltrin proteins, to find out how they may possibly interact with the sperm membranes to control the intracellular signaling that trigger physiological events required for fertilization.

Structural Prediction and In Silico Physicochemical Characterization for Mouse Caltrin I and Bovine Caltrin Proteins.

It is known that caltrin (calcium transport inhibitor) protein binds to sperm cells during ejaculation and inhibits extracellular Ca2+ uptake. Although the sequence and some biological features of mouse caltrin I and bovine caltrin are known, their physicochemical properties and tertiary structure are mainly unknown. We predicted the 3D structures of mouse caltrin I and bovine caltrin by molecular homology modeling and threading. Surface electrostatic potentials and electric fields were calculated using the Poisson–Boltzmann equation. Several different bioinformatics tools and available web servers were used to thoroughly analyze the physicochemical characteristics of both proteins, such as their Kyte and Doolittle hydropathy scores and helical wheel projections. The results presented in this work significantly aid further understanding of the molecular mechanisms of caltrin proteins modulating physiological processes associated with fertilization.

Novel Inhibitory Activity for Serine Protease Inhibitor Kazal Type-3 (Spink3) on Human Recombinant Kallikreins

Kallikrein-related peptidases (KLKs) are trypsin-like and chymotrypsin-like serine proteases which are expressed in several tissues. Their activity is tightly controlled by inhibitors including members of the serine protease Kazal-type (SPINK) family. These enzymes are promising targets for the treatment of skin desquamation, inflammation and cancer. Spink3 or caltrin I is expressed in mouse pancreas and males accessory glands and the resulting mature protein has been associated with different activities such as an inhibitor of trypsin and acrosin activity, calcium transport inhibitor in sperm and inhibitor of cell proliferation during embryogenesis. In this study, we produced a soluble recombinant Spink3 from mouse seminal vesicle (rmSpink3) that inhibited the activity of human KLKs. Using FRET substrates, rmSpink3 exhibited a potent inhibitory activity against human KLK2, KLK3, KLK5 (Ki ranging from 260 to 1500 nM), and to a lesser extent against KLK6, KLK1 and KLK7 (Ki around 3000 nM). As shown by mass spectrometry analysis of rmSpink3 incubated with trypsin, the inhibitor was not truncated by the target enzyme. Based on the in silico analysis of the expression of Spink3/SPINK1 and KLKs it is speculated that some KLKs may be natural targets of Spink3/SPINK1, however experimental confirmation using both proteins from mouse or human origin is needed. This work shows that rmSpink3 is a potent inhibitor of various human KLK members suggesting the potential of this molecule in the diagnosis/prevention of several human diseases.

A Novel Initiation Mechanism of Death in Streptococcus pneumoniae Induced by the Human Milk Protein-Lipid Complex HAMLET and Activated during Physiological Death

To cause colonization or infection, most bacteria grow in biofilms where differentiation and death of subpopulations is critical for optimal survival of the whole population. However, little is known about initiation of bacterial death under physiological conditions. Membrane depolarization has been suggested, but never shown to be involved, due to the difficulty of performing such studies in bacteria and the paucity of information that exists regarding ion transport mechanisms in prokaryotes. In this study, we performed the first extensive investigation of ion transport and membrane depolarization in a bacterial system. We found that HAMLET, a human milk protein-lipid complex, kills Streptococcus pneumoniae (the pneumococcus) in a manner that shares features with activation of physiological death from starvation. Addition of HAMLET to pneumococci dissipated membrane polarity, but depolarization per se was not enough to trigger death. Rather, both HAMLET- and starvation-induced death of pneumococci specifically required a sodium-dependent calcium influx, as shown using calcium and sodium transport inhibitors. This mechanism was verified under low sodium conditions, and in the presence of ionomycin or monensin, which enhanced pneumococcal sensitivity to HAMLET- and starvation-induced death. Pneumococcal death was also inhibited by kinase inhibitors, and indicated the involvement of Ser/Thr kinases in these processes. The importance of this activation mechanism was made evident, as dysregulation and manipulation of physiological death was detrimental to biofilm formation, a hallmark of bacterial colonization. Overall, our findings provide novel information on the role of ion transport during bacterial death, with the potential to uncover future antimicrobial targets.

Pulsing of Membrane Potential in Individual Mitochondria: A Stress-Induced Mechanism to Regulate Respiratory Bioenergetics in Arabidopsis

Mitochondrial ATP synthesis is driven by a membrane potential across the inner mitochondrial membrane; this potential is generated by the proton-pumping electron transport chain. A balance between proton pumping and dissipation of the proton gradient by ATP-synthase is critical to avoid formation of excessive reactive oxygen species due to overreduction of the electron transport chain. Here, we report a mechanism that regulates bioenergetic balance in individual mitochondria: a transient partial depolarization of the inner membrane. Single mitochondria in living Arabidopsis thaliana root cells undergo sporadic rapid cycles of partial dissipation and restoration of membrane potential, as observed by real-time monitoring of the fluorescence of the lipophilic cationic dye tetramethyl rhodamine methyl ester. Pulsing is induced in tissues challenged by high temperature, H(2)O(2), or cadmium. Pulses were coincident with a pronounced transient alkalinization of the matrix and are therefore not caused by uncoupling protein or by the opening of a nonspecific channel, which would lead to matrix acidification. Instead, a pulse is the result of Ca(2+) influx, which was observed coincident with pulsing; moreover, inhibitors of calcium transport reduced pulsing. We propose a role for pulsing as a transient uncoupling mechanism to counteract mitochondrial dysfunction and reactive oxygen species production.

Monitoring of aglycons of yew glycosides (3,5‐dimethoxyphenol, myrtenol and 1‐octen‐3‐ol) as first indicator of yew presence

The toxicity of yew (Taxus spp) is well known from ancient times and is mainly due to taxins acting as inhibitors of calcium and sodium transport across the cell membrane of cardiac myocytes. The confirmation of yew taxins in body fluids can be carried out by liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, before selecting this precise but expensive technique, an orientation test should be done to ascertain yew presence as toxic agent in the organism. As the 3,5-dimethoxyphenol (3,5-DMP), myrtenol and 1-octen-3-ol appear as glycosidically bound volatile compounds and are very yew specific, the detection of 3,5-DMP and the measurement of 1-octen-3-ol / myrtenol concentration ratio constitute reliable indicators of yew presence in forensic cases. The detection of these compounds is easily performed by gas chromatography-mass spectrometry (GC-MS) (SIM) after an enzymatic hydrolysis (β-glucosidase) allowing the release of volatile compounds from yew glycosides.

Phylogeny of whey acidic protein (WAP) four-disulfide core proteins and their role in lower vertebrates and invertebrates

Proteins containing WAP (whey acidic protein) domains with a characteristic WFDC (WAP four-disulfide core) occur not only in mammals (including marsupials and monotremes) but also in birds, reptiles, amphibians and fish. In addition, they are present in numerous invertebrates, from cnidarians to urochordates. Many of those from non-mammalian groups are poorly understood with respect to function or phylogeny. Those well characterized so far are waprins from snakes, perlwapins from bivalves and crustins from decapod crustaceans. Waprins are venom proteins with a single WAP domain at the C-terminus. They display antimicrobial, rather than proteinase inhibitory, activities. Perlwapins, in contrast, possess three WAP domains at the C-terminus and are expressed in the shell nacre of abalones. They participate in shell formation by inhibiting the growth of calcium crystals in the shell. The crustin group is the largest of all WFDC-containing proteins in invertebrates with the vast majority being highly expressed in the haemocytes. Most have a single WAP domain at the C-terminus. The presence and type of the domains between the signal sequence and the C-terminus WAP domain separate the different crustin types. Most of the TypeI and II crustins are antimicrobial towards Gram-positive bacteria, whereas the TypeIII crustins tend to display protease inhibition. Expression studies show that at least some crustins have other important biological effects, as levels change with physiological stress, wound repair, tissue regeneration or ecdysis. Thus WAP domains are widely distributed and highly conserved, serving in diverse physiological processes (proteinase inhibition, bacterial killing or inhibition of calcium transport).

The mode of action of thidiazuron: auxins, indoleamines, and ion channels in the regeneration of Echinacea purpurea L.

The biochemical mechanisms underlying thidiazuron (TDZ)-induced regeneration in plant cells have not been clearly elucidated. Exposure of leaf explants of Echinacea purpurea to a medium containing TDZ results in undifferentiated cell proliferation and differentiated growth as mixed shoot organogenesis and somatic embryogenesis. The current studies were undertaken to determine the potential roles of auxin, indoleamines, and ion signaling in the dedifferentiation and redifferentiation of plant cells. E. purpurea leaf explants were found to contain auxin and the related indoleamine neurotransmitters, melatonin, and serotonin. The levels of these endogenous indoleamines were increased by exposure to TDZ associated with the induction of regeneration. The auxin-transport inhibitor 2,3,5-triiodobenzoic acid and auxin action inhibitor, p-chlorophenoxyisobutyric acid decreased the TDZ-induced regeneration but increased concentrations of endogenous serotonin and melatonin. As well, inhibitors of calcium and sodium transport significantly reduced TDZ-induced morphogenesis while increasing endogenous indoleamine content. These data indicate that TDZ-induced regeneration is the manifestation of a metabolic cascade that includes an initial signaling event, accumulation, and transport of endogenous plant signals such as auxin and melatonin, a system of secondary messengers, and a concurrent stress response.

Direct UV-MALDI-TOF MS Analysis of (Glyco)proteins of Fractions of Bovine Seminal Plasma

Bovine seminal plasma was submitted to chromatography on Con A-Sepharose. The “non-interacting”, “weakly-interacting” and “strongly-interacting” fractions were analyzed through UV-MALDI-TOF MS together with a subfraction of the “non-interacting” material, using sinapinic acid (SA) as matrix. The spectra were obtained in linear positive mode in the 4.0-90.0 kDa mass/charge range showing peaks in well defined zones, namely: 5.5-8.0 kDa, 10.0-12.0 kDa, 12.5-14.0 kDa (major), 23.2-23.7 kDa, 26.1-27.5 kDa and 38.0-40.0 kDa. High sensitivity spectra showed some very small peaks until 90 kDa. Bovine seminal protein (BSP-A3), acidic seminal fluid protein (αSFP) and PDC-109 glycoproteins (BSP-A1 and BSP-A2) were identified. Caltrin, the human epididymis-specific glycoprotein (HE4), the calcium transport inhibitor protein, the inhibitor of metalloprotease 2 (TIMP-2), osteopontin (OPN) and the prostatic acid protease (PAP) were tentatively identified. The molecular weight of some peaks can be arranged in a sequence from that of BSP-A3 going through the molecular weights of glycoforms (including the known BSP-A1 and BSP-A2) which differ in the amounts of neutral hexoses and sialic acids, composing a BSP-family more extended than previously reported. Another two families could be builded up from proteins of molecular weight of about 12730 and 12750 Da and glycoforms which differ from them also by hexoses and sialic acids. The structures of the deduced O-linked oligosaccharides of the glycoforms are in complete agreement to that determined for the BSP-A1 oligosaccharide. Small differences in the m. w. of some (glyco)proteins were attributed to genetic polymorphysm. The identification of proteins and O-linked glycoproteins in the “interacting” fractions of the chromatography suggests that the fractionation was not due to specific affinity interactions but to non-specific hydrophobic interactions of the proteins with the hydrophobic pocked of the Con A.

The influence of sodium–calcium exchange inhibitors on rabbit lens ion balance and transparency

Calcium regulation is essential to the maintenance of lens transparency. To maintain cytoplasmic calcium concentration at the required low level the lens must export calcium continuously. Here, studies were conducted to test whether sodium–calcium exchanger (NCX) inhibitors disturb calcium balance in the rabbit lens. Intact lenses were incubated up to 48 h in the presence or absence of the NCX inhibitor bepridil. Lens sodium, potassium and calcium content were determined by atomic absorption spectrophotometry. Fluo-4 was used to measure epithelial cell cytoplasmic calcium concentration in an intact lens preparation. NCX1 protein expression in lens epithelium was examined by western blot. NCX1 band density was similar in central and equatorial epithelium samples. Lenses exposed to bepridil (30 μM) lost transparency at the anterior and exhibited significant changes in electrolyte and water content. After 48 h, lens calcium content more than doubled, sodium increased four fold and potassium was significantly reduced. In contrast, lenses exposed to inhibitors of reverse mode calcium transport by NCX (KBR7943 or SN-6) remained transparent and the electrolyte and water content of the lens remained unchanged. The ability of bepridil to cause significant changes in lens transparency and electrolyte content points to an important role for NCX-meditated calcium export in the lens.

Thapsigargin Enhances Cell Death in the Gastrointestinal Stromal Tumor Cell Line, GIST-T1, by Treatment with Imatinib (Glivec)

Imatinib is a specific inhibitor of c-KIT that has recently been approved for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumor (GIST). Thapsigargin is an inhibitor of calcium transport to the endoplasmic reticulum (ER) and can inhibit protein maturation. In this study, we evaluated the synergistic cytotoxic effect of thapsigargin and imatinib on the GIST cell line, GIST-T1. Cell viability and cell death were determined by 3,-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. In addition, the amount and activation of c-KIT on the cell surface were measured by flow cytometry and western blot analysis, respectively. Thapsigargin alone (for 5 hr incubation) was shown to decrease the amount of c-KIT on the GIST-T1 cell surface and to slightly inhibit the associated tyrosine kinase activity. Interestingly, thapsigargin significantly enhanced the cell death induced by imatinib compared with the effect of imatinib alone. The results further suggested that thapsigargin induced the increased cell death in GIST-T1 cells co-treated with imatinib, at least in part, via disrupting the translocation of c-KIT to the cell surface, and decreasing the activated c-KIT molecules.

Identification, Evolution, and Regulation of Expression of Guinea Pig Trappin with an Unusually Long Transglutaminase Substrate Domain

Trappins are found in human, bovine, hippopotamus, and members of the pig family, but not in rat and mouse. To clarify the evolution of the trappin genes and the functional significance of their products, we isolated the trappin gene in guinea pig, a species belonging to a rodent family distinct from rat and mouse. Guinea pig trappin was confirmed to encode the same domain structure as trappin, consisting of a signal sequence, an extra large transglutaminase substrate domain, and a whey acidic protein motif. Northern blot analysis and in situ hybridization histochemistry as well as immunohistochemistry demonstrated that guinea pig trappin is expressed solely in the secretory epithelium of the seminal vesicle and that its expression is androgen-dependent. We confirmed that guinea pig trappin is cross-linked by prostate transglutaminase and that the whey acidic protein motif derived from guinea pig trappin has an inhibitory activity against leukocyte elastase. Genome sequence analysis showed that guinea pig trappin belongs to the family of REST (rapidly evolving seminal vesicle transcribed) genes.

Cerebrospinal fluid from human immunodeficiency virus–infected individuals facilitates neurotoxicity by suppressing intracellular calcium recovery

Neurologic decline associated with penetration of human immunodeficiency virus type 1(HIV-1) into the central nervous system is thought to be due, in large part, to inflammation and local secretion of neurotoxic substances. To examine the cellular processes that mediate neurotoxicity in vivo, the authors valuated the ability of neurons to maintain intracellular calcium homeostasis in the presence of toxic cerebrospinal fluid (CSF) (CSF(tox)) collected from a subset of HIV-infected individuals. Exposure of rat neural cultures to CSF(tox) resulted in a gradual increase in intracellular calcium in neurons (+63%), microglia (+251%), and astrocytes (+52%). Pretreatment of neural cultures with CSF(tox) resulted in an exaggerated calcium response to a brief pulse of glutamate and a > 90% suppression of the rate of recovery of intracellular calcium. Attempts to model the deficit using inhibitors of calcium transport across endoplasmic reticulum, mitochondrial, or plasma membrane indicated that blockade of the plasma membrane sodium/calcium exchanger was best able to reproduce the deficits seen during exposure to CSF(tox). Because the inability of cells to maintain calcium homeostasis would lead to exaggerated responses from a wide variety of stimuli, therapeutics designed to facilitate calcium transport from the cell may provide more comprehensive and effective intervention than strategies targeted to specific receptor pathways.

Association of Paracellin-1 with ZO-1 Augments the Reabsorption of Divalent Cations in Renal Epithelial Cells

Paracellin-1 (PCLN-1) belongs to the claudin family of tight junction proteins and possibly plays a critical role in the reabsorption of magnesium and calcium. So far, the physiological properties of PCLN-1 have not been clarified. In the present study, we investigated whether PCLN-1 is associated with ZO-1. We also investigated whether (45)Ca(2+) transport across the paracellular barrier is affected by this association. In vitro binding analysis using glutathione S-transferase fusion protein showed that the C-terminal TRV sequence, especially Thr and Val residues, of PCLN-1 interacts with ZO-1. Next, PCLN-1 was stably expressed in Madin-Darby canine kidney cells using a FLAG tagging vector. ZO-1 was co-immunoprecipitated with the wild-type PCLN-1 and the alanine substitution (TAV) mutant. However, mutants of the deletion (Delta TRV) and the alanine substitution (ARV and TRA) inhibited the association of PCLN-1 with ZO-1. Confocal immunofluorescence demonstrated that the wild-type PCLN-1 and the TAV mutant localized in the tight junction along with ZO-1, but the Delta TRV, ARV, and TRA mutants were widely distributed in the lateral membrane including the tight junction area. Interestingly, monolayers of cells expressing the wild-type PCLN-1 and the TAV mutant showed higher activities of (45)Ca(2+) transport from apical to basal compartments, compared with those expressing the Delta TRV, ARV, and TRA mutants and the mock cells. (45)Ca(2+) transport was inhibited by increased magnesium concentration suggesting that magnesium and calcium were competitively transported by PCLN-1. It was noted that a positive electrical potential gradient enhanced (45)Ca(2+) transport from apical to basal compartments without affecting the opposite direction of transport. Thus, PCLN-1 localizes to the tight junction followed by association with ZO-1, and the PCLN-1.ZO-1 complex may play an essential role in the reabsorption of divalent cations in renal epithelial cells.

Expression of caltrin in the baculovirus system and its purification in high yield and purity by cobalt (II) affinity chromatography

Direct protein extraction from animals is the only approach available to obtain caltrin, calcium transport inhibitor. Here we report the expression and purification of caltrin, previously shown to hinder the influx of calcium into epididymal spermatozoa. Cloning of the caltrin gene into the pCDNA3.1 V5/His-TOPO vector and the subsequent ligation of the caltrin-His sequence into the transfer vector pBacPAK9 allowed the expression of recombinant caltrin using the baculovirus expression vector system (BEVS). Recombinant His-tagged caltrin was purified utilising both nickel (II)–nitrilotriacetic acid (Ni 2+–NTA) and cobalt (II)–carboxymethylaspartate (Co 2+–CmAsp) immobilised metal affinity chromatography (IMAC). Using the BEVS, caltrin-His was identified in the supernatant and in the cell lysate, suggesting that caltrin is a secreted protein. Based on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blot results, purified recombinant caltrin-His was ascertained to be approximately 14.5 kDa. Purification under the Co 2+ system yielded significantly purer protein samples when compared to the Ni 2+ system. Furthermore, Co 2+ was observed to bind the recombinant caltrin-His protein with higher efficiency and specificity and to yield a higher total protein concentration. Collectively, our results indicate that the Co 2+ system would be a better approach for purifying caltrin-His proteins than the Ni 2+.