A bZIP transcription factor (CiFD) regulates drought- and low-temperature-induced flowering by alternative splicing in citrus.

Drought and low temperature are two key environmental factors that induce adult citrus flowering. However, the underlying regulation mechanism is poorly understood. The bZIP transcription factor FD is a key component of the florigen activation complex (FAC) which is composed of FLOWERING LOCUS T (FT), FD, and 14-3-3 proteins. In this study, isolation and characterization of CiFD in citrus found that there was alternative splicing (AS) of CiFD, forming two different proteins (CiFDα and CiFDß). Further investigation found that their expression patterns were similar in different tissues of citrus, but the subcellular localization and transcriptional activity were different. Overexpression of the CiFD DNA sequence (CiFD-DNA), CiFDα, or CiFDß in tobacco and citrus showed early flowering, and CiFD-DNA transgenic plants were the earliest, followed by CiFDß and CiFDα. Interestingly, CiFDα and CiFDß were induced by low temperature and drought, respectively. Further analysis showed that CiFDα can form a FAC complex with CiFT, Ci14-3-3, and then bind to the citrus APETALA1 (CiAP1) promoter and promote its expression. However, CiFDß can directly bind to the CiAP1 promoter independently of CiFT and Ci14-3-3. These results showed that CiFDß can form a more direct and simplified pathway that is independent of the FAC complex to regulate drought-induced flowering through AS. In addition, a bHLH transcription factor (CibHLH96) binds to CiFD promoter and promotes the expression of CiFD under drought condition. Transgenic analysis found that CibHLH96 can promote flowering in transgenic tobacco. These results suggest that CiFD is involved in drought- and low-temperature-induced citrus flowering through different regulatory patterns.

Severe spontaneous acute arterial subdural hematoma as an initial symptom of chronic myeloid leukemia.

Acute subdural hematoma (SDH) is a rare occurrence in chronic myeloid leukemia (CML) patients with only two cases reported in literature. However, sudden severe acute SDH caused by CML has not been reported on. Our patient was admitted for 'sudden unconsciousness for more than 1 hour'. Computed tomography (CT) angiography revealed a large amount of acute SDH on the left side. Physical exam showed the patient's left pupil was dilated and signs of cerebral herniation were present. The preoperative coagulation profile was normal. Emergency craniotomy for hematoma clearance and decompression was performed. During the surgery, a ruptured cerebral artery was located in the perisylvian region and hemostasis was achieved through electrocautery. Pre-operative white blood count was 58,100 cell/µl, with post-operative bone marrow examination、cytogenetic analysis and RT-PCR detection revealing a diagnosis of CML, for which hydroxyurea chemotherapy was initiated. Leukocyte count of the patient gradually returned to normal. After 24 days, the patient regained consciousness and on day 30, repeat CT scan showed no SDH recurrence. The patient recovered with no neurological deficits and achieved a good prognosis.

Central pontine myelinolysis mimicking glioma in diabetes: A case report.

BACKGROUND: Central pontine myelinolysis (CPM) usually occurs during rapid correction of serum osmolality, typically with brainstem lesions presenting uniform signals following enhancement on magnetic resonance imaging (MRI). We report a case of CPM caused by diabetes, which was characterized by glioma-like imaging features and the patient responded well to corticosteroids. CASE SUMMARY: A 49-year-old man with type 2 diabetes was admitted due to numbness and weakness for 6 mo with progressive aggravation for 2 wk. His complete blood count, serum electrolytes, renal and liver function parameters were within the normal range. MRI showed mass lesions in the brainstem, with unusually inhomogeneous signal intensity after contrast-enhanced scans. His symptoms worsened after hypoglycemic therapy. Due to his clinical history and examination results, CPM was considered the most likely diagnosis. Treatment with corticosteroids was administered with a methylprednisolone pulse in the acute phase followed by dose tapering. During the 8-mo follow-up period, his clinical symptoms and imaging features significantly improved. CONCLUSION: Diabetes could rarely be accompanied by CPM, and patients who experience this neurological complication could benefit from corticosteroid treatment. Clinicians should recognize the special relationship between diabetes and CPM, and improve awareness of early identification and appropriate treatment.

Expression of the epigenetic H3K27me3 modifier genes KDM6A and EZH2 in patients with upper tract urothelial carcinoma.

The development of upper tract urothelial carcinoma (UTUC) has been associated with the ingestion of aristolochic acid (AA) in Chinese herbs. The tumors are more malignant and patients have a worse prognosis in China compared with that in Western countries. Recently, whole-genome and exome sequencing of AA-associated UTUCs found that the most frequently mutated gene was lysine demethylase 6A (KDM6A). However, its biological role and clinical significance have not yet been defined in patients with UTUC in China. A total of 108 surgically resected UTUC samples were obtained. Using immunohistochemistry, the protein expression level of KDM6A in the tumors was investigated together with the clinical and pathological characteristics of the patients, including survival times. In the present study, the expression level of KDM6A was significantly lower in UTUC specimens compared with that in samples from the normal urothelium. Lower KDM6A expression was also found to be significantly associated with a higher tumor grade and shorter cancer-specific and disease-free survival times (P=0.023 and P=0.033, respectively). In addition, using immunohistochemical analysis, no positive association was found between KDM6A expression and the expression of H3K27me3 or histone-lysine N-methyltransferase EZH2, a histone methyltransferase that generates H3K27me3. The results of the present study indicated that decreased KDM6A expression level was significantly associated with tumor grade and decreased survival time in UTUC, suggesting that KDM6A expression could be used as a prognostic marker in patients with UTUC in China.

New sesquiterpenoids from the edible mushroom Pleurotus cystidiosus and their inhibitory activity against α-glucosidase and PTP1B.

Nine new sesquiterpenoids, clitocybulol derivatives, clitocybulols G-O (1-9) and three known sesquiterpenoids, clitocybulols C-E (10-12), were isolated from the solid culture of the edible fungus Pleurotus cystidiosus. The structures of compounds 1-12 were determined by spectroscopic methods. The absolute configurations of compounds 1-9 were assigned via the circular dichroism (CD) data analysis. Compounds 1, 6 and 10 showed moderate inhibitory activity against protein tyrosine phosphatase-1B (PTP1B) with IC50 values of 49.5, 38.1 and 36.0µM, respectively.

Sesquiterpenoids with PTP1B Inhibitory Activity and Cytotoxicity from the Edible Mushroom Pleurotus citrinopileatus.

One new perhydrobenzannulated 5,5-spiroketal sesquiterpene, pleurospiroketal F (1), as well as six new modified bisabolene sesquiterpenes pleurotins A-F (2-7) were isolated from solid-state fermentation of Pleurotus citrinopileatus. The structures of compounds 1-7 were determined by NMR and MS spectroscopic analysis. The absolute configuration of 1 was determined by X-ray diffraction analysis, while the absolute configurations of 3-7 were assigned using the in situ dimolybdenum circular dichroism method and circular dichroism data comparison. Protein tyrosine phosphatase 1B plays a crucial role as a negative regulator of the insulin-dependent signal cascades. Therefore, the protein tyrosine phosphatase 1B inhibitor can be used for treating type 2 diabetes mellitus and obesity. Compounds 2 and 6 showed moderate inhibitory effects on protein tyrosine phosphatase 1B with IC50 s of 32.1 µM and 30.5 µM, respectively. The kinetic study confirmed compound 2 to be a noncompetitive inhibitor. Compounds 1-7 did not show cytotoxic activity against cancer cell lines (IC50 > 50 µM).

Fungus ball and emphysematous cystitis secondary to Candida tropicalis: A case report.

Fungus ball and fungal emphysematous cystitis are two rare complications of fungal urinary tract infection. A 53-year-old male patient presented with these complications caused by Candida tropicalis simultaneously. The predisposing factors were diabetes mellitus and usage of broad-spectrum antibiotics. The fungus ball, measuring 3.5 × 2.0 cm on the left wall of the urinary bladder, shrank significantly to 1.6 × 0.8 cm after 5 days of intermittent irrigation with saline before surgery. With transurethral removal of the fungus ball and antifungal treatment with fluconazole, the patient fully recovered. We conclude that a bladder fungus ball and fungal emphysematous cystitis should always be suspected in patients with diabetes mellitus with uncontrolled funguria and abnormal imaging. Treatment should include a systemic antifungal therapy and thorough surgical removal of the fungus ball. A systemic antifungal therapy combined with a local irrigation with saline or antifungal drugs might help decrease the dissemination of fungemia during an invasive manipulation.

Association between the CYP1A1 A2455G polymorphism and risk of cancer: evidence from 272 case-control studies.

A2455G is a common polymorphism in CYP1A1, showing differences in its biological functions. Case-control studies have been performed to elucidate the role of A2455G in cancer; however, the results are conflicting and heterogeneous. Hence, we performed a meta-analysis to investigate the association between cancer susceptibility and A2455G (64,593 cases and 91,056 controls from 272 studies) polymorphism in different inheritance models. We used odds ratios with 95% confidence intervals to assess the strength of the association. Overall, significantly increased cancer risk was observed in any genetic model (dominant model, odds ration [OR] = 1.19, 95% confidence interval [CI] = 1.13-1.25; recessive model: OR = 1.41, 95% CI = 1.29-1.54; additive model: OR = 1.49, 95% CI = 1.35-1.65) when all eligible studies were pooled into the meta-analysis. In further stratified and sensitivity analyses, the elevated risk remained for subgroups of breast cancer, colorectal cancer, esophageal cancer, hepatocellular cancer, head and neck cancer, leukemia, lung cancer, and prostate cancer, but these associations vary in different ethnic populations. In summary, this meta-analysis suggests the participation of A2455G in the susceptibility for some cancers, such as breast cancer, colorectal cancer, lung cancer, and so on. Moreover, ethnicity, histological type of cancer, and smokers seem to contribute to varying expressions of the A2455G on some cancers risk. In addition, our work also points out the importance of new studies for A2455G polymorphism in some cancer types, such as gallbladder cancer, Indians of breast cancer, and Caucasians of ovarians, because these cancer types had high heterogeneity in this meta-analysis (I(2) > 75%).

Effects of apolipoprotein E genotypes on metabolic profile and oxidative stress in southwest Chinese women with polycystic ovary syndrome.

OBJECTIVE: Apolipoprotein (APO) E genetic polymorphism plays an important role in lipid and lipoprotein metabolism, and has been shown to be associated with the risk of metabolic and cardio-cerebrovascular diseases and late-onset Alzheimer's disease. It is not clear, however, whether there are any relationships between the APOE genotypes and PCOS in Chinese women. The aim of this study was to investigate the relationship between APOE genotypes and the risk of polycystic ovary syndrome (PCOS) and to evaluate the effects of the genotypes on metabolic profile and oxidative stress in south-west Chinese women. STUDY DESIGN: A total of 625 patients with PCOS based on the Rotterdam consensus criteria and 514 control women from a population of Chinese Han nationality in the Chengdu area were studied during 2006-2012. APOE genotypes were determined by PCR and restriction fragment length polymorphism analysis. Clinical and metabolic parameters, serum malondialdehyde concentration, and total antioxidant capacity were analyzed. RESULTS: No significant differences were found in the frequencies of APOE genotypes (E2/2, E2/3, E2/4, E3/3, E3/4, E4/4) and alleles (ε2, ε3, ε4) between PCOS and control groups. Compared with ε3 homozygotes (APOE3/3), however, ε2 carriers (APOE2/2+APOE2/3+APOE2/4) had significantly higher body mass index, waist circumference and waist-to-hip ratio, a more adverse glucose and insulin metabolic profile, lower high density lipoprotein (HDL)-cholesterol (C) and APOA1 levels, higher triglyceride (TG)/HDL-C ratio and prevalence of metabolic syndrome (MS), whereas ε4 carriers (APOE3/4+APOE4/4) had higher total cholesterol (TC) and low density lipoprotein (LDL)-C levels in patients with PCOS. CONCLUSIONS: In a cohort of south-west Chinese women, there were no significant associations between any APOE genotype and PCOS. The APOE ε2 allele seems to be related to abdominal obesity, insulin resistance and MS in PCOS women.

Purification and characterization of a novel laccase from the edible mushroom Hericium coralloides.

A novel laccase from the edible mushroom Hericium coralloides was purified by ion exchange chromatography on diethylaminoethyl (DEAE) cellulose, carboxymethyl (CM) cellulose, and Q-Sepharose columns followed by fast protein liquid chromatography gel filtration on a Superdex 75 column. Analysis by gel filtration and SDS-PAGE indicated that the protein is a monomer in solution with a molecular mass of 65 kDa. Its N-terminal amino acid sequence was AVGDDTPQLY, which exhibits partial sequence homology to previously isolated laccases. Optimum activity was observed at pH 2.2 and at 40°C. The enzyme showed activity toward a variety of substrates, the most sensitive of which was 2,2'-azinobis [3-ethylbenzothiazolone-6-sulfonic acid] diammonium salt (ABTS). The degradation activity toward substrates was ABTS > N,N-dimethyl-1,4-phenylenediamine > catechol > 2-methylcatechol > pyrogallol. The laccase did not exert any antiproliferative activity against Hep G2 or MCF 7 tumor cell lines at a concentration of 60 µM, unlike some previously reported mushroom proteins, but showed significant activity toward human immunodeficiency virus-1 (HIV-1) reverse transcriptase with an IC(50) of 0.06 µM.

[Generation and preliminary immunological efficacy of a recombinant human adenovirus-rabies virus glycoprotein].

To construct a recombinant human adenovirus type 5 expressing glycoprotein (GP) of attenuated rabies virus SRV9 and testing immunological efficacy on the immunized mice. Open reading frame of rabies virus GP gene of SRV9 strain was cloned into the shuttle vector of adenovirus expression system in multiple cloning sites to construct the recombinant shuttle plasmid pacAd5 CMV-Gs9, cotransfection was performed into 293AD cells mediated by FuGENE Transfection Reagent with linearized backbone plasmid and recombinant shuttle plasmid, cell cultures were collected after CPE appearance and were identified by PCR and electronmicroscopy, virus titer was measured in 293AD cells. Kunming mice were intraperitoneally injected with 10(6) TCID50 adenovirus, blood for serum preparation was collected through caudal vein pre-immune and post-immune and tested for VNA appearance by fluorescent antibody virus neutralization test (FAVN) detection. Recombinant shuttle plasmid pacAd5 CMV-Gs9 was constructed correctly. A recombinant human adenovirus type 5 was obtained expressing GP protein of rabies virus SRV9. The virus titer reached 10(6) CFU/mL at the least. All mice developed a certain amount of the anti-rabies neutralizing antibody 14 days after intraperitoneal inoculation, while the effective protection rates were 90%. In conclusion, Recombinant adenovirus expressing the rabies virus GP was constructed successfully and a certain amount of neutralizing antibodies were induced in mice, which laid the material foundation for further development of new rabies vaccine.

Predictors and clinical significance of the positive cone margin in cervical intraepithelial neoplasia III patients.

BACKGROUND: Conization is being widely accepted for diagnosis and treatment of cervical intraepithelial neoplasia (CIN). There is controversy as to which factors are most predictive of a positive cone margin and the clinical significance of it. We conducted this study to identify the predictive factors and to evaluate the clinical significance of a positive cone margin in CIN III patients. METHODS: A retrospective review was conducted of 207 patients who had undergone conization due to CIN III from January 2003 to December 2005 at Peking Union Medical College Hospital. Of these, 67 had a subsequent hysterectomy. Univariate and multivariate analysis were utilized to define the predictive factors for a positive cone margin, and to compare the pathologic results of conization with subsequent hysterectomy. RESULTS: One hundred and fifty-one (72.9%) were margin free of CIN I or worse, 37 (17.9%) had CIN lesions close to the margin and 19 (9.2%) had margin involvement. A total of 56 cases (27.1%) had positive cone margins (defined as the presence of CIN at or close to the edge of a cone specimen). Univariate analysis showed that the parity, cytological grade, multi-quadrants of CIN III by punch biopsy, gland involvement, as well as the depth of conization were significant factors correlated with a positive cone margin (P < 0.05). However the age, gravidity, grade of dysplasia in punch biopsy, as well as the cone methods were not significantly correlated (P > 0.05). Multivariate analysis revealed that the cytological grade (OR = 1.92), depth of conization (OR = 2.03), parity (OR = 3.02) and multi-quadrants of CIN III (OR = 4.60) were significant predictors with increased risk for positive margin. The frequency of residual CIN I or worse in hysterectomy specimens was found to be 55.6% (20/36) in patients who were margin free, 71.4% (15/21) in patients with CIN occurring close to margin, and 80.0% (8/10) in patients with margin involvement. The frequency of residual CIN III or worse was found to be 13.9% (5/36), 23.8% (5/21) and 50.0% (5/10) respectively in different groups. CONCLUSIONS: Cytological grade, depth of conization, parity and multi-quadrants of CIN III in punch biopsy were significant factors with increased risk in predicting a positive cone margin. Margin status of conization did not mean the presence or absence of CIN, but rather the varied frequency of residual CIN in specimens of subsequent hysterectomy. In view of this fact, it is suggested that the margin status of conization be a valuable surrogate marker for clinical management of CIN III.

Reversal of P-glycoprotein-mediated multidrug resistance by Alisol B 23-acetate.

Herbal drugs were screened for their activity in reversing multidrug resistance (MDR) in P-glycoprotein (P-gp) over-expressing cancer cells. Through bio-assay guided fractionation an active compound was isolated from Rhizoma Alismatis, the underground part of Alisma orientale and the chemical structure of the isolate compound was confirmed by HPLC, LC-MS and NMR as Alisol B 23-acetate (ABA). ABA restored the sensitivity of MDR cell lines HepG2-DR and K562-DR to anti-tumor agents that have different modes of action but are all P-gp substrates. It restored the activity of vinblastine, a P-gp substrate, in causing G2/M arrest in MDR cells. In a dose-dependent manner, ABA increased doxorubicin accumulation and slowed down the efflux of rhodamin-123 from MDR cells. ABA inhibited the photoaffinity labeling of P-gp by [125I]iodoarylazidoprazosin and stimulated the ATPase activity of P-gp in a concentration-dependent manner, suggesting that it could be a transporter substrate for P-gp. In addition, ABA was also a partial non-competitive inhibitor of P-gp when verapamil was used as a substrate. Our results suggest that ABA may be a potential MDR reversal agent and could serve as a lead compound in the development of novel drugs.

Pyranocoumarin(+/-)-4'-O-acetyl-3'-O-angeloyl-cis-khellactone induces mitochondrial-dependent apoptosis in HL-60 cells.

The pyranocoumarin (+)-4'-O-acetyl-3 'O-angeloyl-cis-khellactone (PC) isolated from Radix Peucedani (root of Peucedanum praeruptorum Dunn) showed a dose-dependent effect at 10 -30 pg/mL on causing apoptotic DNA and nuclear fragmentations in HL-60 cells. After 24 h of PC treatment there were losses of mitochondrial membrane potential and cytochrome c. PC also increased total cellular and mitochondrial Bax protein, stimulated an increase in caspase-dependent Bcl-2 cleavage but showed no effect on Bcl-Xv. These observations strongly suggest activation of the mitochondria apoptotic pathway. The pan-specific caspase inhibitor, ZVAD-fmk, abolished the PC-induced apoptosis,whereas the caspase-8 inhibitor IETD-fmk showed no effect, implying the involvement of the caspase 9 pathway. PC caused a 2 to 12 hour transient increase in phospho-ERK, and a 72 h-long activation of JNK. Pre-treatment with the MEK inhibitor PD98059, which suppresses ERK activation, paradoxically promoted PC-induced mitochondrial cytochrome c release, procaspase-3 and -8 cleavage, and enhanced apoptosis. Our results show that PC triggers mitochondria-mediated apoptosis in HL-60 cells, and the involvement of ERK and JNK signal pathways in the process.

Reversal of multidrug resistance in cancer cells by pyranocoumarins isolated from Radix Peucedani.

The pyranocoumarins, (+/-)-3'-angeloyl-4'-acetoxy-cis-khellactone, were isolated from Radix Peucedani, the dry root of Peucedanum praeruptorum Dunn, through bioassay-guided fractionation. The chemical structure of pyranocoumarins was determined by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. X-ray crystallography showed that there are eight molecules (i.e. two each of four conformers) in each unit cell with their optical activities equally cancelled out. The four conformers are 3'(R)-angeloyl-4'(R)-acetoxy-khellactone in two conformational forms, and 3'(S)-angeloyl-4'(S)-acetoxy-khellactone in two conformational forms. Pyranocoumarins caused apoptotic cell death with IC50 of 41.9+/-2.8 and 17.3+/-8.2 microM for drug-sensitive KB-3-1 and multidrug resistant (MDR) KB-V1, respectively. The two- to threefold sensitivity difference between the two cell lines is interesting considering that the same ratio for doxorubicin is 50-300. Strong synergistic interactions were demonstrated when pyranocoumarins were combined with common anti-tumor drugs including doxorubicin, paclitaxel, puromycin or vincristine in MDR KB-V1 cell line, but not in drug-sensitive KB-3-1 cells. Pyranocoumarins increased doxorubicin accumulation in KB-V1 cells by about 25% after 6 h of incubation. Pyranocoumarins treatment for 24 h down-regulated the expression of P-glycoprotein in KB-V1 cells at both protein and mRNA levels. Pyranocoumarins also transiently reduced the cellular ATP contents in KB-V1 cells in a dose-dependent manner. Our results suggest that pyranocoumarins could be a potential MDR reversing agent.

Pyranocoumarins isolated from Peucedanum praeruptorum as differentiation inducers in human leukemic HL-60 cells.

Differentiation therapy for myeloid leukemia offers great potential as a supplement to the current treatment modalities. In the present report, we investigated if the pyranocoumarins, (+/-)-4'- O-acetyl-3'- O-angeloyl- cis-khellactone (or angular pyranocoumarin, APC) isolated from the medicinal plant Peucedanum praeruptorum Dunn, could induce human acute myeloid leukemic HL-60 cells to differentiate and elucidated the molecular mechanism(s) involved. The ability of HL-60 cells to reduce nitroblue tetrazolium (NBT) was significantly increased after APC treatment for 72 h. In these differentiating HL-60 cells, cell surface differentiation markers CD11b (for myeloid cells) and CD14 (for monocytic cells) were detected in 90.3 % and 70.1 % of the cells, respectively. The differentiation inducing effect of APC was time- and dose-dependent. Treatment with 20 microg/mL APC for 72 h inhibited cell growth by 90 % and cell cycle analysis revealed an increase in the proportion of G1 phase cells. In these growth-inhibited cells the expression of the cyclin-dependent kinase inhibitor p27 kip1, but not p21 WAF1, was up-regulated as shown by Western blotting. Differentiation inducing signal pathways were investigated and it was shown that phospho-MEK and phospho-ERK were elevated shortly after the addition of APC. Pre-incubation of the cells with MEK1 inhibitor PD98059 blocked this APC-induced differentiation. Our results suggest that APC are potent inducers of HL-60 cell differentiation along both the myelocytic and monocytic lineages and are potential agents for differentiation-treatment of leukemia.

Induction of HL-60 cell differentiation by the p38 mitogen-activated protein kinase inhibitor SB203580 is mediated through the extracellular signal-regulated kinase signaling pathway.

The pyridinyl imidazole p38 kinase inhibitor, SB203580, was initially used to block inflammatory cytokine synthesis. Here we report that SB203580 by itself could induce human promyeloid leukemic HL-60 cells to differentiate mainly along the granulocytic lineage, as evidenced by cellular morphological changes, and the concurrent expression of cell surface markers CD11b and CD14. This differentiation induction was time and dose dependent. After 12 h exposure to 10 microM SB203580, 12.5% of the cells became CD11b as compared to only 2.6% in untreated control cells. By 96 h, CD11b cells increased to 72.3%, and among them, 26% were CD14. Morphologically, the cells were smaller in size with lower nuclear/cytoplasmic ratio. The nucleus was indented and nucleoli markedly reduced. However, 10 microM SB203580 had little effect on HL-60 cell growth and survival during the first 72 h, but by 96 h the percentage of cells in G1 phase was markedly increased. These effects of SB203580 were not attributable to its inhibition of p38 kinase activity. Instead, the essential kinases in the extracellular signal-regulated kinase (ERK) pathway such as phospho-Raf-1, phospho-MEK1/2, phospho-ERK1/2 and phospho-p90RSK were all elevated dramatically shortly after cells were exposed to SB203580 and lasted for 24 h before declining. Pre-incubation of cells with 20 microM of PD98059 1 h before addition of SB203580 could completely block the expression of differentiation markers. Our results suggest that SB203580-induced differentiation in HL-60 cells was mediated by activation of MEK/ERK signaling. In conclusion, our data have shown that SB203580 possessed biological activities other than inhibition of p38 and these activities could make it a potential candidate as an inducing agent for cell differentiation in the therapeutic treatment of leukemia.