Enhancing the single-molecule magnetic performance of ß-diketonate Dy(III) complexes by modulating the coordination microenvironment and magnetic interaction: from a mononuclear to a dinuclear structure.

Based on a ß-diketonate ligand, a mononuclear Dy(III) complex, [Dy(dmpd)3(bpy)] (1) (dmpd = 4,4-dimethyl-1-phenylpentane-1,3-dione, bpy = 2,2'-dipyridyl), of [DyN2O6] type has been synthesized with a capping nitrogen-containing coligand. Then, a dual capping coligand 2,2'-bipyrimidine (bmp) is introduced to be a bridge to link two ß-diketonate-Dy(III) motifs, leading to a new dinuclear Dy(III) complex, [Dy2(dmpd)6(bmp)] (2). Dy(III) centers in both complexes feature an N2O6 octacoordinated environment with an approximate square-antiprism geometry (D4d). Without a dc field, the SMM behaviour is absent in complex 1, but can be clearly observed in dinuclear 2 with a Ueff of 87.29 K. The significantly improved magnetism arising in 2 is mainly due to the modulation of the coordination environment around the Dy(III) ions and the superexchange magnetic interactions inside the dinuclear units, thus allowing for the effective inhibition of the quantum tunneling of magnetization at low temperatures and promotion of the uniaxial magnetic anisotropy. For 1, a diamagnetic Y(III) analogue [Y(dmpd)3(bpy)] (3) and diluted sample 1@Y were constructed to further perform the dilution experiment, coupled with theoretical calculations further supporting that the synergetic contributions of intermolecular dipole-dipole interactions, intramolecular coupling and uniaxial magnetic anisotropy cause the enhancement of dynamic magnetic relaxation.

Tumor oxygen microenvironment-tailored electron transfer-type photosensitizers for precise cancer therapy.

The oxygen level in a tumor typically exhibits complex characteristics, ranging from mild hypoxia to moderate and even severe hypoxia. This poses significant challenges for the efficacy of photodynamic therapy, where oxygen is an essential element. Herein, we propose a novel therapeutic strategy and develop a series of lipid droplet-targeting photosensitive dyes (Ser-TPAs), i.e., in situ synergistic activation of two different electron transfer-type reactions. Based on this strategy, Ser-TPAs can synergistically generate O2˙- and nitrogen radicals regardless of the oxygen content, which results in a sustained high concentration of strongly oxidizing substances in the lipid droplets of cancer cells. As such, Ser-TPAs exhibited inhibitory activity against tumor growth in vivo, resulting in a significant reduction in tumor volume (V experimental group : V control group ≈ 0.07). This strategy offers a conceptual framework for the design of innovative photosensitive dyes that are suitable for cancer therapy in complex oxygen environments.

Fluorescence-plane polarization for the real-time monitoring of transferase migration in living cells.

Transferases are enzymes that exhibit multisite migration characteristics. Significantly, enzyme activity undergoes changes during this migration process, which inevitably impacts the physiological function of living organisms and can even lead to related malignant diseases. However, research in this field has been severely hindered by the lack of tools for the simultaneous and differential monitoring of site-specific transferase activity. Herein, we propose a novel strategy that integrates a fluorescence signal response with high sensitivity and an optical rotation signal response with superior spatial resolution. To validate the feasibility of this strategy, transferase γ-glutamyltransferase (GGT) was used as a model system to develop dual-mode chiral probes ACx-GGTB (AC17-GGTB and AC15-GGTB) using chiral amino acids as specific bifunctional recognition groups. The probes undergo structural changes under GGT, resulting in the release of bifunctional recognition groups (chiral amino acids) and simultaneously generate fluorescence signals and optical rotation signals. This dual-mode output exhibits high sensitivity and facilitates differentiation of sites. Furthermore, it enables simultaneous and differential detection of GGT activity at different sites during migration. We anticipate that probes developed based on this strategy will facilitate imaging-based monitoring of the activity for other transferases, thus providing an imaging platform suitable for the real-time tracking of transferase activity changes during migration.

Self-Internal Standard Fluorescence for Ultrasensitive Detecting of mtDNA to Evaluate Matrilineal Genetic Defect Levels.

Mitochondrial DNA (mtDNA) is a unique genetic material characterized by maternal inheritance. It possesses a circular structure devoid of histone protection and exhibits low cellular abundance, which poses great challenges for its sensitive and selective detection at the living cell level. Herein, we have designed three bis-naphthylimide probes with varying linker lengths (NANn-OH, n = 0, 2, 6), facilitating the formation of distinct twisted or folded molecular conformations in the free state. These probes emit the red fluorescence around 627 nm with different fluorescence quantum yields (ΦNAN0-OH = 0.0016, ΦNAN2-OH = 0.0136, and ΦNAN6-OH = 0.0125). When encountering mtDNA (0.4-3.4 µg/mL), these probes undergo conformational changes depending on the length of the attached C-strand and exhibit a gradually increasing fluorescence signal around 453 nm. The fluorescence intensity increased to 13.5-fold, 1.9-fold, and 8.2-fold, respectively. Notably, the red fluorescence intensities around 627 nm remain constant throughout this process, thus serving as an inherent correction mechanism for proportional fluorescence signal enhancement to improve selectivity and sensitivity. NAN0-OH, NAN2-OH, and NAN6-OH showed good linearity for mtDNA in the range of 0.4-3.4 µg/mL with detection limits of LODNAN0-OH = 1.04 µg/mL, LODNAN2-OH = 1.10 µg/mL, and LODNAN6-OH = 1.15 µg/mL. Cellular experiments reveal that NAN6-OH effectively monitors curcumin-induced mtDNA damage in HepG-2 cells while enabling monitoring of genetic mtDNA damage. We anticipate that this tool holds significant potential for the precise evaluation of maternal genetic defects, thereby enhancing hypersensitive assessment in clinical medicine.

Clinical and genetic features in autosomal recessive bestrophinopathy in Chinese cohort.

PURPOSE: To provide a genotype and phenotype characterization of the BEST1 mutation in Chinese patients with autosomal recessive bestrophinopathy (ARB) through multimodal imaging and next-generation sequencing (NGS). METHODS: Seventeen patients from 17 unrelated families of Chinese origin with ARB were included in a retrospective cohort study. Phenotypic characteristics, including anterior segment features, were assessed by multimodal imaging. Multigene panel testing, involving 586 ophthalmic disease-associated genes, and Sanger sequencing were performed to identify disease-causing variants. RESULTS: Among 17 ARB patients, the mean follow-up was 15.65 months and average onset age was 30.53 years (range: 9-68). Best corrected visual acuity ranged from light perception to 0.8. EOG recordings showed a typically decreased Arden ratio in 12 patients, and a normal or slightly decreased Arden ratio in two patients. Anterior features included shallow anterior chambers (16/17), ciliary pronation (16/17), iris bombe (13/17), iridoschisis (2/17), iris plateau (1/17), narrow angles (16/17) and reduced axial lengths (16/17). Sixteen patients had multiple bilateral small, round, yellow vitelliform deposits distributed throughout the posterior pole, surrounding the optic disc. Initial diagnoses included angle-closure glaucoma (four patients), Best disease (three patients), and central serous chorioretinopathy secondary to choroidal neovascularization (CNV) (one patient), with the remainder diagnosed with ARB. Fourteen patients underwent preventive laser peripheral iridotomy, four of whom also received combined trabeculectomy and iridotomy in both eyes for uncontrolled intraocular pressure. One patient received intravitreal conbercept for CNV. Overall, 15 distinct disease-causing variants of BEST1 were identified, with 14 (82.35%) patients having missense mutations. Common mutations included p. Arg255-256 and p. Ala195Val (both 23.68%), with the most frequent sites in exons 7 and 5. CONCLUSIONS: This study provides a comprehensive characterization of anterior segment and genetic features in ARB, with a wide array of morphological abnormalities. Findings are relevant for refining clinical practices and genetic counseling and advancing pathogenesis research.

Photochemical and biological dual-effects enhance the inhibition of photosensitizers for tumour growth.

Photosensitizers typically rely on a singular photochemical reaction to generate reactive oxygen species, which can then inhibit or eradicate lesions. However, photosensitizers often exhibit limited therapeutic efficiency due to their reliance on a single photochemical effect. Herein, we propose a new strategy that integrates the photochemical effect (type-I photochemical effect) with a biological effect (proton sponge effect). To test our strategy, we designed a series of photosensitizers (ZZ-sers) based on the naphthalimide molecule. ZZ-sers incorporate both a p-toluenesulfonyl moiety and weakly basic groups to activate the proton sponge effect while simultaneously strengthening the type-I photochemical effect, resulting in enhanced apoptosis and programmed cell death. Experiments confirmed near-complete eradication of the tumour burden after 14 days (Wlight/Wcontrol ≈ 0.18, W represents the tumour weight). These findings support the notion that the coupling of a type-I photochemical effect with a proton sponge effect can enhance the tumour inhibition by ZZ-sers, even if the basic molecular backbones of the photosensitizers exhibit nearly zero or minimal tumour inhibition ability. We anticipate that this strategy can be generalized to develop additional new photosensitizers with improved therapeutic efficacy while overcoming limitations associated with systems relying solely on single photochemical effects.

Synergistically activated dual-locked fluorescent probes to monitor H2S-induced DNA damage.

Naphthalimide-based fluorescent probes (NAN0-N3 and NAN6-N3) were developed with dual locked fluorescence. Here, ≥1.9 × 10-2 mM of H2S and ≥2.2 × 10-2 µg mL-1 of DNA could unlock a highly sensitive off-on fluorescence response through synergistic changes of the molecular structure and conformation. As such, the probes could monitor DNA damage induced by the overexpression of H2S, and were able to evaluate the degree of apoptosis of living cells mediated by H2S-induced mtDNA or nDNA damage.

Generating Synthesized Ultrasound Biomicroscopy Images from Anterior Segment Optical Coherent Tomography Images by Generative Adversarial Networks for Iridociliary Assessment.

INTRODUCTION: The aim of this study was to investigate the feasibility of generating synthesized ultrasound biomicroscopy (UBM) images from swept-source anterior segment optical coherent tomography (SS-ASOCT) images using a cycle-consistent generative adversarial network framework (CycleGAN) for iridociliary assessment on a cohort presenting for primary angle-closure screening. METHODS: The CycleGAN architecture was adopted to synthesize high-resolution UBM images trained on the SS-ASOCT dataset from the department of ophthalmology, Xinhua Hospital. The performance of the CycleGAN model was further tested in two separate datasets using synthetic UBM images from two different ASOCT modalities (in-distribution and out-of-distribution). We compared the ability of glaucoma specialists to assess the image quality of real and synthetic images. UBM measurements, including anterior chamber, iridociliary parameters, were compared between real and synthetic UBM images. Intra-class correlation coefficients, coefficients of variation, and Bland-Altman plots were used to assess the level of agreement. The Fréchet Inception Distance (FID) was measured to evaluate the quality of the synthetic images. RESULTS: The whole trained dataset included anterior chamber angle images, of which 4037 were obtained by SS-ASOCT and 2206 were obtained by UBM. The image quality of real versus synthetic SS-ASOCT images was similar as assessed by two glaucoma specialists. The Bland-Altman analysis also suggested high consistency between measurements of real and synthetic UBM images. In addition, there was fair to excellent agreement between real and synthetic UBM measurements for the in-distribution dataset (ICC range 0.48-0.97) and the out-of-distribution dataset (ICC range 0.52-0.86). The FID was 21.3 and 24.1 for the synthetic UBM images from the in-distribution and out-of-distribution datasets, respectively. CONCLUSION: We developed a CycleGAN model to translate UBM images from non-contact SS-ASOCT images. The CycleGAN synthetic UBM images showed fair to excellent reproducibility when compared with real UBM images. Our results suggest that the CycleGAN technique is a promising tool to evaluate the iridociliary and anterior chamber in an alternative non-contact method.

mtDNA-Specific Ultrasensitive Near-Infrared Fluorescent Probe Enables the Differentiation of Healthy and Apoptotic Cells.

Mitochondrial DNA (mtDNA) as a class of important genetic material is easily damaged, which can result in a series of metabolic diseases, hereditary disease, and so on. mtDNA is an ultrasensitive indicator for the health of living cells due to the extremely short physiological response time of mtDNA toward damage (ca. 5.0 min). Therefore, the development of specific ultrasensitive fluorescent probes that can in real-time monitor mtDNA in vivo are of great value. With this research, we developed a near-infrared twisted intramolecular charge transfer (TICT) fluorescent probe YON. YON is a thread-like molecule with an A-π-D-π-A structure, based on the dicyanoisophorone fluorophore. The molecular design of YON enabled the specific binding with dsDNA (binding constant (K) = 8.5 × 105 M-1) within 1.3 min. And the appropriate water-oil amphiphilicity makes YON significantly accumulate in the mitochondria, enabling the specific binding to mtDNA. The fluorescence intensity at 640 nm of YON enhanced linearly with increasing concentrations of mtDNA. Dicyanoisophorone as the strong electron-withdrawing group that was introduced into both ends of the molecule resulted in YON being a classic quadrupole, so it could ultrasensitively detect trace mtDNA. The minimum detection limit was 71 ng/mL. Moreover, the large Stokes shift (λex = 435 nm, λem = 640 nm) makes YON suitable for "interference-free" imaging of mtDNA. Therefore, YON was used to monitor trace changes of mtDNA in living cells; more importantly, it could be used to evaluate the health of cells by monitoring microchanges of mtDNA, enabling the ultrasensitive evaluation of apoptosis.

Dual-factor Synergistically Activated ESIPT-based Probe: Differential Fluorescence Signals to Simultaneously Detect α-Naphthyl Acetate and Acid α-Naphthyl Acetate Esterase.

α-Naphthyl acetate esterase (α-NAE) and acid α-naphthyl acetate esterase (ANAE), a class of special esterases, are important for lymphocyte typing and immunocompetence-monitoring. As such, the simultaneous detection of α-NAE and ANAE has become a target to effectively improve the accuracy in lymphocyte typing. Therefore, we developed a dual-factor synergistically activated ESIPT-based probe (HBT-NA) to detect α-NAE and ANAE sensitively, rapidly, and simultaneously in a differential manner. HBT-NA exhibits differential fluorescence signal outputs toward small changes of α-NAE and ANAE activities. HBT-NA displays a weak fluorescence signal at 392 nm over a pH range from 6.0 to 7.4. However, when it interacts with α-NAE (0-25 U) at pH = 7.4, the fluorescence intensity at 392 nm enhanced linearly within 60 s (F392 nm/F0392 nm = 0.042 Cα-NAE + 1.1, R2 = 0.99). Furthermore, HBT-NA emits ratiometric fluorescence signals (F505 nm/F392 nm) for ANAE (0-25 U) at pH = 6.0 within 2.0 min, exhibiting a good linear relationship (F505 nm/F392 nm = 0.83CANAE - 1.75, R2 = 0.99). The differential fluorescence signals can be used to simultaneously detect the activities of α-NAE and ANAE in solutions and complex living organisms. More importantly, based on the differential fluorescence signals toward α-NAE and ANAE, T lymphocytes and B lymphocytes could be successfully typed and differentiated among nontyped lymphocytes, facilitating the real-time evaluation of their immune functions using flow cytometry. Hence, HBT-NA could be used for the ultrasensitive detection of the enzyme activities of α-NAE and ANAE, the real-time precise typing of lymphocytes, and the monitoring of immunocompetence.

A2AR Antagonists Upregulate Expression of GS and GLAST in Rat Hypoxia Model.

BACKGROUND: The aim of this study was to research the effects of glutamine synthetase (GS) and glutamate aspartate transporter (GLAST) in rat Müller cells and the effects of an adenosine A2AR antagonist (SCH 442416) on GS and GLAST in hypoxia both in vivo and in vitro. METHODS: This study used RT-PCR and Western blotting to quantify the expressions of GS and GLAST under different hypoxic conditions as well as the expressions of GS and GLAST at different drug concentrations. A cell viability assay was used to assess drug toxicity. RESULTS: mRNA and protein expression of GS and GLAST in hypoxia Group 24 h was significantly increased. mRNA and protein expressions of GS and GLAST both increased in Group 1 µM SCH 442416 compared with other groups. One micromolar SCH 442416 could upregulate GS and GLAST's activity in hypoxia both in vivo and in vitro. CONCLUSIONS: Hypoxia activates GS and GLAST in rat retinal Müller cells in a short time in vitro. (2) A2AR antagonists upregulate the activity of GS and GLAST in hypoxia both in vivo and in vitro.

A molecular-logic gate for COX-2 and NAT based on conformational and structural changes: visualizing the progression of liver disease.

Lighting up the relevant lesion boundaries during operations is vital for guiding the effective resection of hepatopathic tissue. We envisioned that molecular-logic gates, which are known for their excellent digital correlation between input and output signals, could be used to facilitate differential visualization of lesion boundaries. Herein, a series of flexible molecules, naphthalene imide-indole derivatives (IAN) were prepared and evaluated as molecular-logic gates. The input and output signals of the IAN derivatives were successfully used to highlight different hepatopathic regions in order to facilitate boundary differentiation. The IAN derivatives produce different signals due to collaborative changes in the conformation and structure. The hepatopathy-related enzymes (COX-2 and NAT) were used to induce conformational and structural changes in IAN derivatives. Based on these enzyme induced synergistic effects, IAN can sensitively emit different coloured signals such as green, cyan and blue (output signals) as a function of the different input signals, i.e. the different activity of COX-2 and NAT in solution and living cells. Significantly, the IAN derivatives were successfully used to distinguish the boundaries of hepatopathic lesions in tissues after spraying with IAN derivatives (mild cirrhosis, severe cirrhosis, in addition to early and late hepatocellular carcinoma) under a hand held lamp at 365 nm by naked eye.

Light-activated "cycle-reversible intramolecular charge transfer" fluorescent probe: monitoring of pHi trace change induced by UV light in programmed cell death.

Under the synergistic effects of protonation and deprotonation, a light-activated fluorescent probe (UV-SP) exhibited "cycle-reversible intramolecular charge transfer (ICT)" for different pH after activation by UV light, resulting in emission of multiple ratio fluorescent signals (FI563/FI595 and FI664/FI595). Based on these kinds of response signals, UV-SP can specifically monitor the cycle-reversible trace change of intracellular pH caused by UV radiation. More importantly, according to the stable and invariant multiple ratio fluorescent signals, UV-SP can sort cells entering programmed death.

A dynamic invertible intramolecular charge-transfer fluorescence probe: real-time monitoring of mitochondrial ATPase activity.

A dynamic invertible intramolecular charge-transfer (ICT) process could provide abundant response signals for real-time monitoring in living organisms. Herein, based on dynamic invertible ICT, we have reported a cancer cell-targeted fluorescence probe (OPM) for mitochondrial ATPase activity. Due to its abundant response signals, OPM could real-time monitor mitochondrial ATPase activity during the cancer apoptosis process, successfully.

[Ecological function and application of toxin beta-ODAP in grass pea (Lathyrus sativus)].

Grass pea (Lathyrus sativus) is a legume with various adverse adaptability and rich nutrition. However, it can lead to the human and animal neurotoxicity after long-term consumption due to its neurotoxin, beta-N-oxalyl-L-alpha, beta-diaminopropionic acid (beta-ODAP), limiting its utilization. This paper summarized the influences of beta-ODAP on osmotic adjustment and growth regulation in grass pea under drought stress, the research progress in analysis methods, toxicological mechanisms and practical utility of beta-ODAP, and the breeding strategies for low- and zero-beta-ODAP. Beta-ODAP synthesis was found to be abundant in grass pea under drought stress and its content was enhanced gradually with the increasing extent of drought stress. beta-ODAP could supply nitrogen for plant growth and seed development, scavenge reactive oxygen species (ROS), involve in osmotic adjustment as a soluble amino acid, transport zinc-ions as a carrier molecule, and impact nodule development. However, increasing the content of sulfur-containing amino acids (methionine and cysteine) could decrease the level of toxicity of grass pea. There were a lot of investigations on collecting genetic resources, cross breeding, tissue culture, and gene manipulation for low- and zero-toxin in grass pea in recent years. Although beta-ODAP could induce excitotoxicity by damaging intracellular Ca2+ homeostasis and as glutamate analogues, it has medicinal value on hemostasis and anti-tumor.

Abscisic acid promotes accumulation of toxin ODAP in relation to free spermine level in grass pea seedlings (Lathyrus sativus L.).

Interrelationship among abscisic acid (ABA) content, accumulation of free polyamines and biosynthesis of beta-N-oxalyl-l-alpha,beta-diaminopropionic acid (ODAP) was studied in grass pea (Lathyrus sativus L.) seedlings under drought stress induced by 10% polyethylene glycol (PEG6000). Increase of ABA content occurred prior to that of ODAP and polyamine contents, and was found significantly positive correlation between ABA content and ODAP content. Addition of exogenous ABA increased ODAP content in leaves. On the other hand, pretreatment with alpha-difluoromethylarginine (DFMA), a polyamine biosynthesis inhibitor, significantly suppressed the accumulation of free putrescine (Put), free spermidine (Spd) and free spermine (Spm), which in turn inhibited biosynthesis of ODAP in well-watered leaves. Meanwhile, addition of exogenous Put alleviated DFMA-induced inhibition on the biosynthesis of Put and Spd, but did not affect the biosynthesis of Spm and ODAP in well-watered leaves. Same result was also achieved in drought-stressed leaves. Increasing accumulation of ODAP was significantly correlated with increasing Spm content (R=0.7957**) but not with that of Spd and Put. Therefore, it can be argued that ABA stimulated the biosynthesis of ODAP simultaneously with increasing the level of free Spm under drought stress condition.

Lathyrus sativus (grass pea) and its neurotoxin ODAP.

Lathyrus sativus (grass pea) is a high-yielding, drought-resistant legume consumed as a food in Northern India and neighboring countries as well as in Ethiopia. Its development into an important food legume, however, has been hindered by the presence of the neurotoxin - beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (beta-ODAP) in seeds which, if consumed in large quantities for prolonged periods, can cause irreversible paralysis. Recently, some low-toxin lines have been developed that may prove safe for both animal and human foods. Cultivation of L. sativus should thus be considered in suitable regions because the demand for legume animal feed protein products is expected to increase. This paper addresses advances in understanding L. sativus from the perspective of its taxonomy, genetics, ecology, chemistry, nutrition, medicine, biology and for animal nutrition.

[Role of exogenous Ca2+ in the somatic embryogenesis of Lycium barbarum L].

In this study, Embryogenic and non-embryogenic calli were separately obtained by cultivation of leaf segments on MS medium containing and not containing 2,4-D 0.2 mg/L. The calli were transferred to an 2,4-D-free MS medium containing different concentrations of (45)Ca(2+) and EGTA cultured, and microscopic examination of tissue sliced, gamma-ray energy spectrum analysis, ELISA and two-dimensional polyacrylamide-gel electrophoresis were used to study the relation between changes in Ca(2+) concentration and protein composition changes during somatic embryogenesis. The results showed that: (1) Calli of dedifferentiation were obtained by cultivating in the inductive medium (MS+2,4-D 0.2 mg/L) and then transferred to the 2,4-D-free (MS) differentiation medium. After cultivating, the large number of embryogenic cells divided and somatic embryogenic calli (EC) were formed; embryogenic cell differentiation and somatic embryo ware not formed when the dedifferentiation calli, which were cultivated in the inductive medium without 2,4-D, ware transferred to the cultivating of differentiation, so calli were called non-embryogenic calli (NEC). (2) SE frequency of EC was rised with exogenous Ca(2+) concentration was going up, and adding peak value (70.5% to 74.5%) when Ca(2+)concentration was from 0.8 to 1.6 mmol/L, then SE frequency was dropped markedly with Ca(2+) concentration was farther increasing. Formation of meristematic cell aggregates of NEC was also enhanced when exogenous Ca(2+) concentration was from 0.8 to 1.6 mmol/L. (3) After adding EGTA, which was Ca(2+) antagonist, SE frequency was dropped markedly, and SE frequency was fallen along with increased of EGTA concentration. When EGTA concentration went up to 1.2 micromol/L, SE frequency dropped to 5%, and the formation of meristematic cell aggregates on NEC was inhibited. (4) When 2 microCi (45)Ca(2+)/mL was added, the uptake of (45)Ca(2+) by EC and NEC was different, two uptake peaks of (45)Ca(2+)appeared in EC at the embryogenic cell differentiation of stage, and the uptake of (45)Ca(2+) of EC was 4-5 times higher than that of NEC, and the uptake frequency of (45)Ca(2+) improved from 54.1% to 74.5%. The uptake of (45)Ca(2+) by NEC during development not only was lower than that by EC but also there were no such marked peak as those with EC. (5) The CaM content examined by ELISA was increased markedly at multi-cellular embryo and globular embryoid stage of EC. After adding Ca(2+), the CaM content increased significantly, the CaM content of EC was 2-3 times that of NEC. (6) The IEF/SDS-PAGE results showed that the numbers and amount of protein components were widely different between the two kinds of callus with different morphogenesis patterns, the number of proteins of EC had more components than those of NEC. The largest differences protein species presented with Ca(2+) ware added, the more proteins presented on the range of molecular weight was from 43 kD to 66 kD and pI values was from 4.0 to 7.0.