[Application of high content imaging technique in research on effective components of traditional Chinese medicine: a review].

High content imaging(HCI) technique that combines automatic high throughput with high-resolution cell imaging, is characterized by abundant data information, high imaging sensitivity, easy visualization and standardization, and is commonly used in the cellular(or subcellular) phenotypic analysis. Abundant phenotypic information can be obtained by using HCI in one experiment, including cellular morphology, cellular structure, and signal transduction pathways of related functions, on the basis of the maintenance of the integrity of cellular structures and functions. Multiple studies have shown that a series of dynamic spatio-temporal interactive change processes were induced by the disturbance of cells by specific factors, making cell phenotypes change accordingly, especially for the slight perturbation response of cells. Generally, the detection of one or several endpoint effect indicators is often difficult to accurately and comprehensively reflect the overall efficacy information of traditional Chinese medicine(TCM) because of its unique characteristics of multi-components and multi-targets. The application of HCI is thus helpful to discover the effective components and their action modes in the complex system of TCM. This paper reviewed the application progress in the HCI technique in the screening of active components and their regulation mechanism to provide references for further research.

Quantitative proteomics reveals arsenic attenuates stem-loop binding protein stability via a chaperone complex containing heat shock proteins and ERp44.

Arsenic pollution impacts health of millions of people in the world. Inorganic arsenic is a carcinogenic agent in skin and lung cancers. The stem-loop binding protein (SLBP) binds to the stem-loop of the canonical histone mRNA and regulates its metabolism during cell cycle. Our previous work has shown arsenic induces ubiquitin-proteasome dependent degradation of SLBP and contributes to lung cancer. In this study, we established the first comprehensive SLBP interaction network by affinity purification-mass spectrometry (AP-MS) analysis, and further demonstrated arsenic enhanced the association between SLBP and a crucial chaperone complex containing heat shock proteins (HSPs) and ERp44. Strikingly, knockdown of these proteins markedly rescued the protein level of SLBP under arsenic exposure conditions, and abolished the increasing migration capacity of BEAS-2B cells induced by arsenic. Taken together, our study provides a potential new mechanism that a chaperone complex containing HSPs and ERp44 attenuates the stability of SLBP under both normal and arsenic exposure conditions, which could be essential for arsenic-induced high cell migration.

ImmunoPET imaging of multiple myeloma with [68Ga]Ga-NOTA-Nb1053.

PURPOSE: Multiple myeloma (MM) remains incurable and its diagnosis relies heavily on bone marrow aspiration and biopsy. CD38 is a glycoprotein highly specific for MM. Antibody therapeutics (e.g., daratumumab) targeting CD38 have shown encouraging efficacy in treating MM, either as a monotherapy agent or in combination with other regimens. However, efficient stratification of patients who might benefit from daratumumab therapy and timely monitoring of the therapeutic responses are still clinical challenges. This work aims to devise a CD38-targeted imaging strategy and assess its value in diagnosing MMs. METHODS: By labeling a CD38-specific single domain antibody (Nb1053) with 68Ga (t1/2 = 1.1 h), we developed a CD38-targeted immuno-positron emission tomography (immunoPET) imaging probe [68Ga]Ga-NOTA-Nb1053. The probe was developed with good radiochemical yield (> 50%), excellent radiochemical purity (> 99%), and immunoreactivity (> 95%). The diagnostic accuracy of the probe was thoroughly investigated in preclinical MM models. RESULTS: ImmunoPET imaging with [68Ga]Ga-NOTA-Nb1053 specifically depicted all the subcutaneous and orthotopic MM lesions, outperforming the traditional 18F-fluorodeoxyglucose PET and the nonspecific [68Ga]Ga-NOTA-NbGFP immunoPET. More importantly, daratumumab preloading significantly reduced [68Ga]Ga-NOTA-Nb1053 uptake in the disseminated bone lesions, indicating the overlapping targeting epitopes of [68Ga]Ga-NOTA-Nb1053 with that of daratumumab. Furthermore, premedication with sodium maleate or fructose significantly decreased kidney retention of [68Ga]Ga-NOTA-Nb1053 and improved the diagnostic value of the probe in lymphoma models. CONCLUSION: This work successfully developed a novel CD38-targeted immunoPET imaging approach that enabled precise visualization of CD38 and diagnosis of MMs. Upon clinical translation, [68Ga]Ga-NOTA-Nb1053 immunoPET may serve as a valuable CD38-targeted molecular imaging toolbox, facilitating early diagnosis of MM and precise assessment of the therapeutic responses.

Resolving the topological enigma in Ca2+ signaling by cyclic ADP-ribose and NAADP.

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are two structurally distinct messengers that mobilize the endoplasmic and endolysosomal Ca2+ stores, respectively. Both are synthesized by the CD38 molecule (CD38), which has long been thought to be a type II membrane protein whose catalytic domain, intriguingly, faces to the outside of the cell. Accordingly, for more than 20 years, it has remained unresolved how CD38 can use cytosolic substrates such as NAD and NADP to produce messengers that target intracellular Ca2+ stores. The discovery of type III CD38, whose catalytic domain faces the cytosol, has now begun to clarify this topological conundrum. This article reviews the ideas and clues leading to the discovery of the type III CD38; highlights an innovative approach for uncovering its natural existence; and discusses the regulators of its activity, folding, and degradation. We also review the compartmentalization of cADPR and NAADP biogenesis. We further discuss the possible mechanisms that promote type III CD38 expression and appraise a proposal of a Ca2+-signaling mechanism based on substrate limitation and product translocation. The surprising finding of another enzyme that produces cADPR and NAADP, sterile α and TIR motif-containing 1 (SARM1), is described. SARM1 regulates axonal degeneration and has no sequence similarity with CD38 but can catalyze the same set of multireactions and has the same cytosolic orientation as the type III CD38. The intriguing finding that SARM1 is activated by nicotinamide mononucleotide to produce cADPR and NAADP suggests that it may function as a regulated Ca2+-signaling enzyme like CD38.

A cytosolic chaperone complex controls folding and degradation of type III CD38.

Cluster of differentiation 38 (CD38) is the best-studied enzyme catalyzing the synthesis of the Ca2+ messenger cyclic ADP-ribose. It is a single-pass transmembrane protein, but possesses dual orientations. We have documented the natural existence of type III CD38 in cells and shown that it is regulated by a cytosolic activator, calcium- and integrin-binding 1 (CIB1). However, how type III CD38 can be folded correctly in the reductive cytosol has not been addressed. Using the yeast two-hybrid technique with CD38's catalytic domain (sCD38) as bait, here we identified a chaperone, Hsp70-interacting protein (Hip), that specifically interacts with both the type III CD38 and sCD38. Immunoprecipitation coupled with MS identified a chaperone complex associated specifically with sCD38. Pharmacological and siRNA-mediated knockdown of Hsp90 chaperones decreased the expression levels of both sCD38 and type III CD38, suggesting that these chaperones facilitate their folding. Moreover, knockdown of Hsc70 or DNAJA2 increased the levels of both CD38 types, consistent with the roles of these proteins in mediating CD38 degradation. Notably, Hip knockdown decreased type III CD38 substantially, but only marginally affected sCD38, indicating that Hip was selective for the former. More remarkably, DNAJA1 knockdown decreased sCD38 but increased type III CD38 levels. Mechanistically, we show that Hsc70 mediates lysosomal degradation of type III CD38, requiring the lysosomal receptor Lamp2A and the C19-motif in the C terminus of CD38. Our results indicate that folding and degradation of type III CD38 is effectively controlled in cells, providing further strong support of its physiological relevance.

The transferrin receptor CD71 regulates type II CD38, revealing tight topological compartmentalization of intracellular cyclic ADP-ribose production.

The CD38 molecule (CD38) catalyzes biogenesis of the calcium-mobilizing messenger cyclic ADP-ribose (cADPR). CD38 has dual membrane orientations, and type III CD38, with its catalytic domain facing the cytosol, has low abundance but is efficient in cyclizing cytosolic NAD to produce cADPR. The role of cell surface type II CD38 in cellular cADPR production is unknown. Here we modulated type II CD38 expression and assessed the effects of this modulation on cADPR levels. We developed a photoactivatable cross-linking probe based on a CD38 nanobody, and, combining it with MS analysis, we discovered that cell surface CD38 interacts with CD71. CD71 knockdown increased CD38 levels, and CD38 knockout reciprocally increased CD71, and both could be cocapped and coimmunoprecipitated. We constructed a chimera comprising the N-terminal segment of CD71 and a CD38 nanobody to mimic CD71's ligand property. Overexpression of this chimera induced a dramatically large decrease in CD38 via lysosomes. Remarkably, cellular cADPR levels did not decrease correspondingly. Bafilomycin-mediated blockade of lysosomal degradation greatly elevated active type II CD38 by trapping it in the lysosomes but also did not increase cADPR levels. Retention of type II CD38 in the endoplasmic reticulum (ER) by expressing an ER construct that prevented its transport to the cell surface likewise did not change cADPR levels. These results provide first and direct evidence that cADPR biogenesis occurs in the cytosol and is catalyzed mainly by type III CD38 and that type II CD38, compartmentalized in the ER or lysosomes or on the cell surface, contributes only minimally to cADPR biogenesis.

Cytosolic interaction of type III human CD38 with CIB1 modulates cellular cyclic ADP-ribose levels.

CD38 catalyzes the synthesis of the Ca2+ messenger, cyclic ADP-ribose (cADPR). It is generally considered to be a type II protein with the catalytic domain facing outside. How it can catalyze the synthesis of intracellular cADPR that targets the endoplasmic Ca2+ stores has not been resolved. We have proposed that CD38 can also exist in an opposite type III orientation with its catalytic domain facing the cytosol. Here, we developed a method using specific nanobodies to immunotarget two different epitopes simultaneously on the catalytic domain of the type III CD38 and firmly established that it is naturally occurring in human multiple myeloma cells. Because type III CD38 is topologically amenable to cytosolic regulation, we used yeast-two-hybrid screening to identify cytosolic Ca2+ and integrin-binding protein 1 (CIB1), as its interacting partner. The results from immunoprecipitation, ELISA, and bimolecular fluorescence complementation confirmed that CIB1 binds specifically to the catalytic domain of CD38, in vivo and in vitro. Mutational studies established that the N terminus of CIB1 is the interacting domain. Using shRNA to knock down and Cas9/guide RNA to knock out CIB1, a direct correlation between the cellular cADPR and CIB1 levels was demonstrated. The results indicate that the type III CD38 is functionally active in producing cellular cADPR and that the activity is specifically modulated through interaction with cytosolic CIB1.

CD38 produces nicotinic acid adenosine dinucleotide phosphate in the lysosome.

Nicotinic acid adenosine dinucleotide phosphate (NAADP) is a Ca2+-mobilizing second messenger that regulates a wide range of biological activities. However, the mechanism of its biogenesis remains controversial. CD38 is the only enzyme known to catalyze NAADP synthesis from NADP and nicotinic acid. CD38-mediated catalysis requires an acidic pH, suggesting that NAADP may be produced in acidic endolysosomes, but this hypothesis is untested. In this study, using human cell lines, we specifically directed CD38 to the endolysosomal system and assessed cellular NAADP production. First, we found that nanobodies targeting various epitopes on the C-terminal domain of CD38 could bind to cell surface-localized CD38 and induce its endocytosis. We also found that CD38 internalization occurred via a clathrin-dependent pathway, delivered CD38 to the endolysosome, and elevated intracellular NAADP levels. We also created a CD38 variant for lysosome-specific expression, which not only withstood the degradative environment in the lysosome, but was also much more active than WT CD38 in elevating cellular NAADP levels. Supplementing CD38-expressing cells with nicotinic acid substantially increased cellular NAADP levels. These results demonstrate that endolysosomal CD38 can produce NAADP in human cells. They further suggest that CD38's compartmentalization to the lysosome may allow for its regulation via substrate access, rather than enzyme activation, thereby providing a reliable mechanism for regulating cellular NAADP production.

Solution-phase total synthesis of teixobactin.

The first solution-phase total synthesis of the cyclic depsipeptide teixobactin is described. Stereoselective construction of l-allo-enduracididine was established, and the protective groups for the peptide coupling reactions and conditions for the assembly of the fragments were also optimised. The longest linear sequence for the total synthesis was 20 steps from the known l-cis-4-hydroxyproline derivative and gave a 5.6% overall yield. This solution-phase total synthesis could serve as a complement to the current solid-phase synthesis of teixobactin.

Anti-Multiple Myeloma Activity of Nanobody-Based Anti-CD38 Chimeric Antigen Receptor T Cells.

Chimeric antigen receptor T cells (CAR-Ts) are a promising strategy for the treatment of many cancers, including multiple myeloma (MM), a hematological malignancy characterized by the high expression of CD38. To broaden the applications of using CD38 as a therapeutic target for the disease, we developed a new nanobody against CD38 and constructed a CD38-CAR that was composed of this nanobody as the targeting domain, and 4-1BB and CD3ζ as the costimulatory and activating domains, in a lentiviral vector. CD3+ T cells from healthy individuals were transduced with the CD38-CAR at an efficiency higher than 60%, as determined by CD38-CAR expression using flow cytometry. The CD38-CAR-Ts proliferated efficiently and produced more inflammatory cytokines, such as IL-2, IFN-γ, and TNF-α, when activated. The CD38-CAR-Ts effectively lysed CD38+ MM cell lines, including LP-1, RPMI 8226, OPM2, and MOLP8, and primary MM cells from multiple myeloma patients. The specificity was demonstrated by the fact that CD38-CAR-Ts showed little cytotoxicity on LP-1 cells with CD38 knocked out or on K562 cells, which do not express CD38. CD38-CAR-Ts appeared to have a very slight cytotoxicity against CD38+ fractions of T cells, B cells, and natural killer cells. In addition, the lysis of CD34+ hematopoietic progenitor cells did not completely inhibit the development of colony-forming units. In vivo, CD38-CAR-Ts inhibited tumor growth in NOD/SCID mice that were subcutaneously inoculated with RPMI 8226 cells. These results demonstrate that the CD38-CAR-Ts constructed with the anti-CD38 nanobody are a promising approach for the treatment of multiple myeloma.

[Changes of ion absorption, distribution and essential oil components of flowering Schizonepeta tenuifolia under salt stress].

In this paper, a pot experiment using quartz sands was conducted to study the effects of different concentrations of NaCl (0, 25, 50, 75, 100 mmol·L⁻¹) on the ion absorption, distribution and essential oil components of flowering Schizonepeta tenuifolia. The results showed that as NaCl concentration increased, Na⁺ content of root, stem, leaf and flower increased significantly, and that of the aerial parts was in a higher level than in the root. Regarding the K⁺ content, it decreased in the root but increased in stem, leaf and flower. Some changes were detected in the Ca²âº content, but not significant on the whole. The value of K⁺/Na⁺ and Ca²âº/Na⁺ reduced as a result of increasing NaCl concentrations. The content of essential oil increased under medium salt treatment (50 mmol·L⁻¹ NaCl). However, the synthesis and accumulation of essential oil was inhibited by the serious salt treatment (100 mmol·L⁻¹ NaCl). Over 98% of the essential oil components were terpenes, in which pulegone and menthone were the most two abundant compounds. Varieties of essential oil components did not change significantly under salt stress but their relative proportions did. The transformation of pulegone to menthone was enhanced and the value of pulegone/menthone based on their relative contents decreased with NaCl concentration increasing. Consequently, menthone ranked the most abundant compound by replacing pulegone. Relative content of D-limonene increased under medium and serious salt stress, and that of ß-caryophyllene only increased under mild treatments. So our research could provide references for the standard cultivation on saline soil of S. tenuifolia.

Luteolinidin Protects the Postischemic Heart through CD38 Inhibition with Preservation of NAD(P)(H).

We recently showed that ischemia/reperfusion (I/R) of the heart causes CD38 activation with resultant depletion of the cardiac NADP(H) pool, which is most marked in the endothelium. This NADP(H) depletion was shown to limit the production of nitric oxide by endothelial nitric oxide synthase (eNOS), which requires NADPH for nitric oxide production, resulting in greatly altered endothelial function. Therefore, intervention with CD38 inhibitors could reverse postischemic eNOS-mediated endothelial dysfunction. Here, we evaluated the potency of the CD38 inhibitor luteolinidin, an anthocyanidin, at blocking CD38 activity and preserving endothelial and myocardial function in the postischemic heart. Initially, we characterized luteolinidin as a CD38 inhibitor in vitro to determine its potency and mechanism of inhibition. We then tested luteolinidin in the ex vivo isolated heart model, where we determined luteolinidin uptake with aqueous and liposomal delivery methods. Optimal delivery methods were then further tested to determine the effect of luteolinidin on postischemic NAD(P)(H) and tetrahydrobiopterin levels. Finally, through nitric oxide synthase-dependent coronary flow and left ventricular functional measurements, we evaluated the efficacy of luteolinidin to protect vascular and contractile function, respectively, after I/R. With enhanced postischemic preservation of NADPH and tetrahydrobiopterin, there was a dose-dependent effect of luteolinidin on increasing recovery of endothelium-dependent vasodilatory function, as well as enhancing the recovery of left ventricular contractile function with increased myocardial salvage. Thus, luteolinidin is a potent CD38 inhibitor that protects the heart against I/R injury with preservation of eNOS function and prevention of endothelial dysfunction.

Determinants of the membrane orientation of a calcium signaling enzyme CD38.

CD38 catalyzes the synthesis of two structurally distinct messengers for Ca²âº-mobilization, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), from cytosolic substrates, NAD and NADP, respectively. CD38 is generally thought of as a type II membrane protein with its catalytic site facing outside. We recently showed that CD38 exists, instead, in two opposite membrane orientations. The determinant for the membrane topology is unknown. Here, specific antibodies against type III CD38 were designed and produced. We show that mutating the positively charged residues in the N-terminal tail of CD38 converted its orientation to type III, with the catalytic domain facing the cytosol and it was fully active in producing intracellular cADPR. Changing the serine residues to aspartate, which is functionally equivalent to phosphorylation, had a similar effect. The mutated CD38 was expressed intracellularly and was un-glycosylated. The membrane topology could also be modulated by changing the highly conserved di-cysteine. The results indicate that the net charge of the N-terminal segment is important in determining the membrane topology of CD38 and that the type III orientation can be a functional form of CD38 for Ca²âº-signaling. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

[The synthesis of purine derivatives and its inhibitory activity on CD38 NADase].

CD38 is a multifunctional enzyme expressed in a variety of mammalian tissues, its catalytic activity was involved in a wide range of physiological processes. Based on the reported inhibitor of human CD38 NADase, 33 purine derivatives were designed and synthesized. The biological activity assay showed that compounds 20 and 38 exhibited almost the same extent of inhibitory activities on human CD38 NADase as the lead compound H2. The results also revealed that small substituents at C-6 of purine ring gave no obvious effect on inhibitory activity, but phenylpropionyl moiety at N-2 could affect the binding mode of the compound with CD38. This study provides a reliable basis for future rational design of inhibitors for CD38.

Design, synthesis and SAR studies of NAD analogues as potent inhibitors towards CD38 NADase.

Nicotinamide adenine dinucleotide (NAD), one of the most important coenzymes in the cells, is a substrate of the signaling enzyme CD38, by which NAD is converted to a second messenger, cyclic ADP-ribose, which releases calcium from intracellular calcium stores. Starting with 2'-deoxy-2'-fluoroarabinosyl-ß-nicotinamide adenine dinucleotide (ara-F NAD), a series of NAD analogues were synthesized and their activities to inhibit CD38 NAD glycohydrolase (NADase) were evaluated. The adenosine-modified analogues showed potent inhibitory activities, among which 2'-deoxy-2'-fluoroarabinosyl-ß-nicotinamide guanine dinucleotide (ara-F NGD) was the most effective one. The structure-activity relationship of NAD analogues was also discussed.

A novel fluorescent cell membrane-permeable caged cyclic ADP-ribose analogue.

Cyclic adenosine diphosphate ribose is an endogenous Ca(2+) mobilizer involved in diverse cellular processes. A cell membrane-permeable cyclic adenosine diphosphate ribose analogue, cyclic inosine diphosphoribose ether (cIDPRE), can induce Ca(2+) increase in intact human Jurkat T-lymphocytes. Here we synthesized a coumarin-caged analogue of cIDPRE (Co-i-cIDPRE), aiming to have a precisely temporal and spatial control of bioactive cIDPRE release inside the cell using UV uncaging. We showed that Co-i-cIDPRE accumulated inside Jurkat cells quickly and efficiently. Uncaging of Co-i-cIDPRE evoked Ca(2+) release from endoplasmic reticulum, with concomitant Ca(2+) influx in Jurkat cells. Ca(2+) release evoked by uncaged Co-i-cIDPRE was blocked by knockdown of ryanodine receptors (RyRs) 2 and 3 in Jurkat cells. The associated Ca(2+) influx, on the other hand, was abolished by double knockdown of Stim1 and TRPM2 in Jurkat cells. Furthermore, Ca(2+) release or influx evoked by uncaged Co-i-cIDPRE was recapitulated in HEK293 cells that overexpress RyRs or TRPM2, respectively, but not in wild-type cells lacking these channels. In summary, our results indicate that uncaging of Co-i-cIDPRE incites Ca(2+) release from endoplasmic reticulum via RyRs and triggers Ca(2+) influx via TRPM2.

Base-Exchange Enabling the Visualization of SARM1 Activities in Sciatic Nerve-Injured Mice.

Enzymes are important in homeostasis in living organisms. Since abnormal enzyme activities are highly associated with many human diseases, detection of in vivo activities of a specific enzyme is important to study the pathology of the related diseases. In this work, we have designed and synthesized a series of new small-molecule-activatable fluorescent probes for the imaging of Sterile Alpha and TIR Motif-containing 1 (SARM1) activities based on its transglycosidase activities (base-exchange reactions of NAD+). Probe 1a was found to undergo base-exchange reactions with NAD+ in the presence of activated SARM1 but not CD38 nor NADase and formed a highly emissive product AD-1a [about a 100-fold fluorescence enhancement in 20 min with a 150 nm (5665 cm-1) Stokes shift and a 100 nm (3812 cm-1) red shift]. This probe exhibited a higher reactivity and sensitivity than those commonly used for SARM1 imaging. The utilities of 1a have also been demonstrated in live-cell imaging and detection of in vivo activities of SARM1 in a sciatic nerve injury mouse model.

Pathogenic mechanism of Eimeria tenella autophagy activation of chicken embryo cecal epithelial cells induced by Eimeria tenella.

Eimeria tenella mainly invades and develops into cecal epithelial cells of chickens, resulting in cecal epithelial cell damage. Infectious intracellular pathogens possibly act by influencing the autophagy process after invading cells. The interaction between E. tenella and the autophagy of host cells was explored by infecting E. tenella with chick embryo cecal epithelial cells. Transmission electron microscopy, laser confocal microscopy, and Western blot analysis were used to demonstrate that E. tenella infection could induce autophagy in host cells. Results showed that infection with E. tenella induced the formation of autophagosomes in cells. The expression of ATG 5, Beclin-1, and LC3B-II proteins were significantly (P < 0.01) increased after E. tenella infected host cells. Expression of p62 protein levels were significantly (P < 0.01) decreased in host cells infected with E. tenella. Chloroquine (CQ) significantly (P < 0.01) increased the expression levels of LC3B-II and P62 in E. tenella-infected host cells. Rapamycin (RAPA) induced autophagy in host cells, thus reducing the intracellular infection of E. tenella. By contrast, the infection rate of E. tenella increased in cells treated with 3-Methyladenine (3-MA). Hence, E. tenella sporozoite infection could induce autophagy activation in chick embryo cecal epithelial cells, and enhanced autophagy could reduce the infection rate of E. tenella.

Cytosolic CD38 protein forms intact disulfides and is active in elevating intracellular cyclic ADP-ribose.

CD38 catalyzes the synthesis of cyclic ADP-ribose (cADPR), a Ca(2+) messenger responsible for regulating a wide range of physiological functions. It is generally regarded as an ectoenzyme, but its intracellular localization has also been well documented. It is not known if internal CD38 is enzymatically active and contributes to the Ca(2+) signaling function. In this study, we engineered a novel soluble form of CD38 that can be efficiently expressed in the cytosol and use cytosolic NAD as a substrate to produce cADPR intracellularly. The activity of the engineered CD38 could be decreased by mutating the catalytic residue Glu-226 and increased by the double mutation E146A/T221F, which increased its cADPR synthesis activity by >11-fold. Remarkably, the engineered CD38 exhibited the ability to form the critical disulfide linkages required for its enzymatic activity. This was verified by using a monoclonal antibody generated against a critical disulfide, Cys-254-Cys-275. The specificity of the antibody was established by x-ray crystallography and site-directed mutagenesis. The engineered CD38 is thus a novel example challenging the general belief that cytosolic proteins do not possess disulfides. As a further refinement of this approach, the engineered CD38 was placed under the control of tetracycline using an autoregulated construct. This study has set the stage for in vivo manipulation of cADPR metabolism.