Enhanced therapeutic potential of antibody fragment via IEDDA-mediated site-specific albumin conjugation.

BACKGROUND: The use of single-chain variable fragments (scFvs) for treating human diseases, such as cancer and immune system disorders, has attracted significant attention. However, a critical drawback of scFv is its extremely short serum half-life, which limits its therapeutic potential. Thus, there is a critical need to prolong the serum half-life of the scFv for clinical applications. One promising serum half-life extender for therapeutic proteins is human serum albumin (HSA), which is the most abundant protein in human serum, known to have an exceptionally long serum half-life. However, conjugating a macromolecular half-life extender to a small protein, such as scFv, often results in a significant loss of its critical properties. RESULTS: In this study, we conjugated the HSA to a permissive site of scFv to improve pharmacokinetic profiles. To ensure minimal damage to the antigen-binding capacity of scFv upon HSA conjugation, we employed a site-specific conjugation approach using a heterobifunctional crosslinker that facilitates thiol-maleimide reaction and inverse electron-demand Diels-Alder reaction (IEDDA). As a model protein, we selected 4D5scFv, derived from trastuzumab, a therapeutic antibody used in human epithermal growth factor 2 (HER2)-positive breast cancer treatment. We introduced a phenylalanine analog containing a very reactive tetrazine group (frTet) at conjugation site candidates predicted by computational methods. Using the linker TCO-PEG4-MAL, a single HSA molecule was site-specifically conjugated to the 4D5scFv (4D5scFv-HSA). The 4D5scFv-HSA conjugate exhibited HER2 binding affinity comparable to that of unmodified 4D5scFv. Furthermore, in pharmacokinetic profile in mice, the serum half-life of 4D5scFv-HSA was approximately 12 h, which is 85 times longer than that of 4D5scFv. CONCLUSIONS: The antigen binding results and pharmacokinetic profile of 4D5scFv-HSA demonstrate that the site-specifically albumin-conjugated scFv retained its binding affinity with a prolonged serum half-life. In conclusion, we developed an effective strategy to prepare site-specifically albumin-conjugated 4D5scFv, which can have versatile clinical applications with improved efficacy.

Photobiomodulation Can Enhance Stem Cell Viability in Cochlea with Auditory Neuropathy but Does Not Restore Hearing.

Sensorineural hearing loss is very difficult to treat. Currently, one of the techniques used for hearing rehabilitation is a cochlear implant that can transform sound into electrical signals instead of inner ear hair cells. However, the prognosis remains very poor if sufficient auditory nerve cells are not secured. In this study, the effect of mouse embryonic stem cells (mESC) and photobiomodulation (PBM) combined treatment on auditory function and auditory nerve cells in a secondary neuropathy animal model was investigated. To confirm the engraftment of stem cells in vitro, cochlear explants were treated with kanamycin (KM) to mimic nerve damage and then cocultured with GFP-mESC. GFP-mESCs were observed to have attached and integrated into the explanted samples. An animal model for secondary neurodegeneration was achieved by KM treatment and was treated by a combination therapy of GFP-mESC and NIR-PBM at 8 weeks of KM treatment. Hearing recovery by functional testing using auditory brain stem response (ABR) and eABR was measured as well as morphological changes and epifluorescence analysis were conducted after 2 weeks of combination therapy. KM treatment elevated the hearing threshold at 70-80 dB and even after the combination treatment with GFP-mESC and PBM was applied, the auditory function was not restored. In addition, the stem cells transplanted into cochlea has exponentially increased due to PBM treatment although did not produce any malignancy. This study confirmed that the combined treatment with mESC and PBM could not improve hearing or increase the response of the auditory nerve. Nevertheless, it is noteworthy in this study that the cells are distributed in most cochlear tissues and the proliferation of stem cells was very active in animals irradiated with PBM compared to other groups wherein the stem cells had disappeared immediately after transplantation or existed for only a short period of time.

Discordant renal progression of Fabry disease in male monozygotic twins: a case report.

Background: Fabry disease (FD) is a rare X-linked lysosomal storage disease caused by mutations in the GLA gene that encodes α-galactosidase A (α-GAL). Clinical phenotypes tend to vary in monozygotic female twins because mutations are located on the X-chromosome, whereas similar phenotypes are found in male monozygotic twins. Here we report the case of male monozygotic twins with FD presenting with distinguishable renal phenotypes. Case: A 49-year-old male patient who visited the hospital with proteinuria 14 years prior was readmitted for the same issue. His monozygotic twin brother had started hemodialysis 6 months prior due to renal failure of unknown origin. The patient's renal function was within the normal range, while his spot urine protein-to-creatinine ratio was 557 mg/g. Echocardiography revealed left ventricular hypertrophy (LVH). The findings of a renal biopsy were consistent with FD. Genetic testing identified a c.656T>C mutation in the GLA gene, and α-GAL activity was significantly decreased. Genetic screening of his family clarified that his mother, older sister, twin brother, and his daughter had the same genetic mutations. The patient received enzyme replacement therapy 34 times. Subsequently, migalastat was initiated that continues today. Renal function and proteinuria remain stable, and the LVH has mildly improved. Conclusion: This is the first case of male monozygotic twins expressing different progressions of FD. Our findings demonstrate the possibility that environmental or epigenetic factors may critically influence genotype-phenotype discordance.

Polymer-Infiltrated Metal-Organic Frameworks for Thin-Film Composite Mixed-Matrix Membranes with High Gas Separation Properties.

Thin-film composite mixed-matrix membranes (TFC-MMMs) have potential applications in practical gas separation processes because of their high permeance (gas flux) and gas selectivity. In this study, we fabricated a high-performance TFC-MMM based on a rubbery comb copolymer, i.e., poly(2-[3-(2H-benzotriazol-2-yl)-4-hydroxyphenyl] ethyl methacrylate)-co-poly(oxyethylene methacrylate) (PBE), and metal-organic framework MOF-808 nanoparticles. The rubbery copolymer penetrates through the pores of MOF-808, thereby tuning the pore size. In addition, the rubbery copolymer forms a defect-free interfacial morphology with polymer-infiltrated MOF-808 nanoparticles. Consequently, TFC-MMMs (thickness = 350 nm) can be successfully prepared even with a high loading of MOF-808. As polymer-infiltrated MOF is incorporated into the polymer matrix, the PBE/MOF-808 membrane exhibits a significantly higher CO2 permeance (1069 GPU) and CO2/N2 selectivity (52.7) than that of the pristine PBE membrane (CO2 permeance = 431 GPU and CO2/N2 selectivity = 36.2). Therefore, the approach considered in this study is suitable for fabricating high-performance thin-film composite membranes via polymer infiltration into MOF pores.

High-Loading Poly(ethylene glycol)-Blended Poly(acrylic acid) Membranes for CO2 Separation.

Poly(ethylene glycol) (PEG) is an amorphous material of interest owing to its high CO2 affinity and potential usage in CO2 separation applications. However, amorphous PEG often has a low molecular weight, making it challenging to form into the membrane. The crystalline high average molar mass poly(ethylene oxide) (PEO) cannot exhibit CO2 separation characteristics. Thus, it is crucial to employ low molecular weight PEG in high molecular weight polymers to increase the CO2 affinity for CO2 separation membranes. In this work, poly(acrylic acid) (PAA)/PEG blend membranes with a PEG-rich phase were simply fabricated by physical mixing with an ethanol solvent. The carbonyl group of the PAA and the hydroxyl group of the PEG formed a hydrogen bond. Furthermore, the thermal stability, glass transition temperature, and surface hydrophilicity of PAA/PEG blend membranes with various PEG concentrations were further characterized. The PAA/PEG(1:9) blend membrane exhibited an improved CO2 permeability of 51 Barrer with high selectivities relative to the other gas species (H2, N2, and CH4; CO2/H2 = 6, CO2/N2 = 63, CO2/CH4 = 21) at 35 °C and 150 psi owing to the enhanced CO2 affinity with the amorphous PEG-rich phase. These PAA/PEG blend membrane permeation characteristics indicate a promising prospect for CO2 capture applications.

Technical note: Multi-receiver combination method for phase-based electrical property tomography of the breast.

BACKGROUND: Phase-based electrical property tomography (EPT) is a technique that allows conductivity reconstruction with only phase of the B1 field under the assumption that the magnitude of the B1 fields are homogeneous. The more this assumption is violated, the less accurate the reconstructed conductivity. Thus, a method that ensures homogeneity of | B 1 - | $| {{\rm{B}}_1^ - } |$ field is important for breast image using multi-receiver coil. PURPOSE: To develop a method for multi-receiver combination for phase-based EPT usable for breast EPT imaging in the clinic. METHODS: Theory of the proposed method is presented. To validate the proposed method, the phantom and in-vivo experiments were conducted. Conductivity images were reconstructed using the transceive phase of the combined image and results were compared with another combination method. RESULTS: The proposed method's conductivity results were more stable than those of the previous method when | B 1 + | $| {{\rm{B}}_1^ + } |$ was not homogeneous and when the homogeneous contrast region was small. The phantom and in-vivo results indicate that the proposed method produces improved conductivity images than the previous method. The proposed combination method also increased the conductivity contrast between benign and cancerous tissues. CONCLUSION: The proposed method produced more stable multi-receiver combination for phase-based EPT of the breast in a clinical environment.

Comparison of the Clinical Effectiveness Between Infrared Thermography and Electrophysiology Tests in Spinal Intradural Extramedullary Schwannoma.

Objective: Subjective pain is experienced differently by each patient; therefore, modalities that can objectify subjective symptoms are useful. Electrophysiology tests and infrared (IR) thermography can present subjective symptoms in an objective manner. This study aimed to compare the effectiveness of electrophysiology tests and IR thermography in patients with intradural extramedullary (IDEM) schwannoma and statistically analyze the results to verify the positive relationship between the subjective neurologic symptoms and test results. Methods: We retrospectively analyzed the data from 23 patients, pathologically confirmed to have IDEM spinal schwannoma after surgery between January 2012 and December 2020. All patients were preoperatively examined using IR thermography and an electrophysiology test. IR thermography was conducted again week after operation. The IR thermography results were classified as either positive or negative. Results: Radiculopathy symptoms were reported in 16 cases and myelopathy in 7 cases. Among the radiculopathy patients, 9 out of 16 (56.2%) showed positive electrophysiology test results. Among the myelopathy patients, 2 out of 7 (28.5%) showed positive electrophysiology test results. In the radiculopathy group, 15 out of 16 (93.7%) patients showed positive IR thermography results. In the myelopathy group, 2 out of 7 (28.5%) patients showed positive IR thermography results. The correlation between the IR thermography and electrophysiology test was analyzed. In the radiculopathy group, positive electrophysiology test result was obtained in 8 out of 15 (53.5%) patients with positive IR thermography result. Conclusion: In patients with IDEM schwannoma presenting radiculopathy symptoms, IR thermography is a complementary tool to objectify the neurological symptoms.

Computation-Aided Design of Albumin Affibody-Inserted Antibody Fragment for the Prolonged Serum Half-Life.

Single-chain variable fragments (scFvs) have been recognized as promising agents in cancer therapy. However, short serum half-life of scFvs often limits clinical application. Fusion to albumin affibody (ABD) is an effective and convenient half-life extension strategy. Although one terminus of scFv is available for fusion of ABD, it is also frequently used for fusion of useful moieties such as small functional proteins, cytokines, or antibodies. Herein, we investigated the internal linker region for ABD fusion instead of terminal region, which was rarely explored before. We constructed two internally ABD-inserted anti-HER2 4D5scFv (4D5-ABD) variants, which have short (4D5-S-ABD) and long (4D5-L-ABD) linker length respectively. The model structures of these 4D5scFv and 4D5-ABD variants predicted using the deep learning-based protein structure prediction program (AlphaFold2) revealed high similarity to either the original 4D5scFv or the ABD structure, implying that the functionality would be retained. Designed 4D5-ABD variants were expressed in the bacterial expression system and characterized. Both 4D5-ABD variants showed anti-HER2 binding affinity comparable with 4D5scFv. Binding affinity of both 4D5-ABD variants against albumin was also comparable. In a pharmacokinetic study in mice, the 4D5-ABD variants showed a significantly prolonged half-life of 34 h, 114 times longer than that of 4D5scFv. In conclusion, we have developed a versatile scFv platform with enhanced pharmacokinetic profiles with an aid of deep learning-based structure prediction.

Desmoid Fibromatosis in the Multifidus Muscle Misdiagnosed as Intramuscular Schwannoma by Incisional Biopsy: A Case Report.

Desmoid fibromatosis is a locally aggressive myofibroblastic neoplasm. In this study, we report a case of desmoid fibromatosis in the paraspinal muscle that was misdiagnosed as intramuscular schwannoma through incisional biopsy at another hospital. We performed total excision of the mass lesion with a clear margin. We found that for an accurate diagnosis, magnetic resonance imaging, incisional biopsy and excisional biopsy were required.

Exposure to long-term evolution radiofrequency electromagnetic fields decreases neuroblastoma cell proliferation via Akt/mTOR-mediated cellular senescence.

The aim of this study was to examine the potential effects of long-term evolution (LTE) radiofrequency electromagnetic fields (RF-EMF) on cell proliferation using SH-SY5Y neuronal cells. The growth rate and proliferation of SH-SY5Y cells were significantly decreased upon exposure to 1760 MHz RF-EMF at 4 W/kg specific absorption rate (SAR) for 4 hr/day for 4 days. Cell cycle analysis indicated that the cell cycle was delayed in the G0/G1 phase after RF-EMF exposure. However, DNA damage or apoptosis was not involved in the reduced cellular proliferation following RF-EMF exposure because the expression levels of histone H2A.X at Ser139 (γH2AX) were not markedly altered and the apoptotic pathway was not activated. However, SH-SY5Y cells exposed to RF-EMF exhibited a significant elevation in Akt and mTOR phosphorylation levels. In addition, the total amount of p53 and phosphorylated-p53 was significantly increased. Data suggested that Akt/mTOR-mediated cellular senescence led to p53 activation via stimulation of the mTOR pathway in SH-SY5Y cells. The transcriptional activation of p53 led to a rise in expression of cyclin-dependent kinase (CDK) inhibitors p21 and p27. Further, subsequent inhibition of CDK2 and CDK4 produced a fall in phosphorylated retinoblastoma (pRb at Ser807/811), which decreased cell proliferation. Taken together, these data suggest that exposure to RF-EMF might induce Akt/mTOR-mediated cellular senescence, which may delay the cell cycle without triggering DNA damage in SH-SY5Y neuroblastoma cells.

Clinical Implications of Poloxamer 407 as Packing Material in an Animal Model.

BACKGROUND: Endoscopic ear surgery has recently increased, but it is still inconvenient and time-consuming to place packing material in the middle ear with one hand. Poloxamer 407 (P407) is a thermo-reversible gel that can be easily administered with one hand into the middle ear cavity in liquid form. Upon warming to body temperature, the gel form of P407 can support the graft in the target position and is known to prevent postsurgical tissue adhesion. OBJECTIVES: We aim to investigate the feasibility of P407 as packing material in an animal model. Male Hartley guinea pigs (350 and 400 g) were utilized in this study. METHOD: The animals were randomly divided into 3 groups according to the packing material: the control group, the P407 group, and the gelatin group. To assess the role of packing material on bacterial colonization, left ears were inoculated with Streptococcus pneumoniae through the tympanic membrane using a 0° endoscope. Five days after inoculation, the middle ear cavity was packed through a transbullar approach using 18% P407 or gelatin in both ears. In the control group, no ear pack was inserted. The tympanic membrane was examined every week using a 0° 1.9-mm endoscope until 6 weeks. Half of the animals in each group were sacrificed 6 weeks after placement of the packing materials. RESULTS: Compared with the absorbable gelatin sponge, the P407 group showed little inflammation or fibrosis in the tympanic membrane and middle ear mucosa regardless of bacterial inoculation. The gelatin group showed severe otorrhea or perforation until 2 weeks in the right ear (2 of 4) and the left ear (1 of 4). Even though the endoscopic findings were similar between both packing groups at 6 weeks, histological analysis showed persistent packing material, inflammatory cells, and fibrosis in the gelatin group compared to the P407 group. CONCLUSIONS: This study suggested that P407 is feasible as a packing material to handle with one hand and to prevent adhesion, especially in infected middle ear mucosa. Although there is a lack of data on how well P407 supports grafts, we suggest that P407 could be a candidate for packing material in endoscopic ear surgery.

Early exposure to radiofrequency electromagnetic fields at 1850 MHz affects auditory circuits in early postnatal mice.

In the present study, we measured the spontaneous post synaptic currents (sPSCs) at the post synaptic principle cells of the medial nucleus of the trapezoid body (MNTB) in early postnatal mice after exposure to 1850 MHz radiofrequency electromagnetic fields (RF-EMF). sPSC frequencies and amplitudes were significantly increased in the RF-EMF exposed group. Moreover, the number of synaptic vesicles in the calyx of Held was significantly increased in presynaptic nerve terminals. Following RF-EMF exposure, the number of docking synaptic vesicles in the active zone increased, thereby expanding the total length of the presynaptic active zone in the calyx of Held. These data suggest that the increased sPSCs are a result of greater synaptic vesicle release from presynaptic nerves. However, we found no morphological changes in the inner hair cell ribbon synapses. Further, there were no significant changes in the hearing threshold of the auditory brainstem response at postnatal day 15. Our results indicate that exposure to RF-EMF at an early postnatal stage might directly affect brainstem auditory circuits, but it does not seem to alter general sound perception.

Photobiomodulation by laser therapy rescued auditory neuropathy induced by ouabain.

Auditory neuropathy is a hearing disorder caused by impaired auditory nerve function. The lack of information about the pathophysiology of this disease limits early diagnosis and further treatment. Laser therapy is a novel approach to enhance nerve growth or induce axonal regeneration. We induced auditory neural degeneration sparing the sensory epithelium with local ouabain application in an animal model and observed the rescue effect of photobiomodulation (PBM), showing recovered auditory function and favorable histologic outcome. Hearing was evaluated using the auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE). Seven days after ouabain application, the animals were sacrificed to evaluate the morphological changes. DPOAE change was not observed in all groups after ouabain application indicating no changes of outer hair cell function. Ouabain application increased the ABR thresholds increase, while the use of ouabain plus laser produced lower threshold compared to the ouabain group. Hematoxylin and Eosin staining of cochlea mid-modiolar sections in animals treated with ouabain showed damaged spiral ganglion cells, neurofilaments, and post synaptic puncta. Ouabain plus laser group showed higher number of spiral ganglion cells, higher density of neurofilaments, and higher number post synaptic puncta counts compared with ouabain application group. Short-term application of ouabain caused spiral ganglion cell damage while sparing the inner and outer hair cells in gerbils. Photobiomodulation alleviated the hearing loss caused by ouabain induced auditory neuropathy. The results indicate the possible role of photobiomodulation therapy for inner ear diseases accompanied by spiral ganglion degeneration.

Protein/peptide based nanomaterials for energy application.

Biological systems have developed unique capabilities to generate, harness and store energy in efficient ways. In recent years, biologically inspired approaches have been introduced as a new approach to find breakthroughs for next generation energy devices and storage. Of particular interest are efforts to translate biological principles directly into synthetic energy systems. In this review, we focus on the use of proteins and protein mimicry for energy applications. We highlight the major advances and results achieved with proteins as new concept energy devices.

Prognosis of patients with pT1b/T2 gallbladder carcinoma who have undergone laparoscopic cholecystectomy as an initial operation.

BACKGROUNDS/AIMS: Laparoscopic cholecystectomy (LC) has become a standard procedure for treatment of benign gallbladder diseases. There has been a small proportion of gallbladder cancer (GBC) which was incidentally found in the gallbladder specimen, and LC has been tried in some patients with faintly suspected GBC. This study intended to analyze the prognosis of patients with pT1b/T2 GBC who have undergone LC and the outcome of extended re-operation. METHODS: After analyzing the institutional profiles of 500 GBC patients who have undergone surgical resection, we selected 64 patients who underwent LC initially from January 1996 to December 2008 and whose gallbladder pathology was confined to pT1b or pT2 lesions. Of them, 34 patients (53.1%) underwent extended reoperation. Their medical records were reviewed retrospectively. RESULTS: In the LC only group (n=30), mean age of the 16 pT1 patients was 65.7±12.5 years and mean age of the 14 pT2 patients was 66.7±10.1 years. In the reoperation group (n=34), mean age of the 8 pT1b patients was 52.6±9.9 years and in 26 pT2 patients, mean age was 59.2±7.9 years. The reoperation group showed a younger patient age pattern than the LC only group (p=0.001). The types of reoperation were liver resection with lymph node (LN) dissection in 17, bile duct resection with LN dissection in 2, and hepatectomy and bile duct resection with LN dissection in 15. In the LC only group, the 5-year survival rate (5-YSR) was 70.3% in pT1b and 43.2% in pT2. In the reoperation group, 5-YSR was 62.5% in pT1b (n=8) and 59.5% in pT2 (n=26). A survival comparison between the two groups showed no significant survival gain in pT1 patients (p=0.69) and in pT2 patients (p=0.14). In our whole database analysis, 5-YSR of pT1bNx lesions was 70% after cholecystectomy and 78% after extended cholecystectomy. Lymph node metastasis was identified in 11% of pT1b lesions. For pT2N0 lesions, overall 5-YSR was 62% after R0 resection, showing no survival difference between primary extended surgery and LC-redo operation (p=0.45). CONCLUSIONS: The survival gain of reoperation was not evident in pT1b lesions. In contrast, some noticeable but not statistically significant survival difference was observed in pT2 lesions. Thus, reoperation for pT1b/T2 GBC following LC is indicated for individualized reasons, especially in patients with pT1b lesions. Old age was one of the important factors in deciding not to reoperate.

Glutamine (Q)-peptide screening for transglutaminase reaction using mRNA display.

Information on subsite specificity of the transglutaminase (TG) is important to design any specific peptides for TG's applications and inhibitor studies. Here, mRNA display was introduced for identifying the subsite specificity of TG from Streptomyces mobaraensis (STG). Functionally active peptides expressed from mRNA display library were differentially conjugated to hexa lysine (K6-beads according to their relative activities for STG. The active peptide substrates for STG were enriched through six rounds of screening, and its corresponding cDNA/mRNA sequences were identified by DNA sequencing. The results showed that tripeptides such as LQQ and TQP do not show any activity for STG, but the minimum size of the peptide displaying STG activity is pentapeptide. One such predicted peptide sequence, that is, RLQQP (TQ1), showed higher reactivity (ca. 182% conjugation yield) to STG than that of the highly active sequence, that is, control-Q (PQPQLPYPQPQLPY), well-known previously for mammalian TG2. Furthermore, when recombinant DsRed was tagged with TQ1 sequence at its C-terminal, DsRed-TQ1 underwent efficient covalent-immobilization onto alginate-gelatin bead by STG reaction, showing a Q-peptide application as a useful tagging molecule.

Biologically inspired strategy for programmed assembly of viral building blocks with controlled dimensions.

Facile fabrication of building blocks with precisely controlled dimensions is imperative in the development of functional devices and materials. We demonstrate the assembly of nanoscale viral building blocks of controlled lengths using a biologically motivated strategy. To achieve this we exploit the simple self-assembly mechanism of Tobacco mosaic virus (TMV), whose length is solely governed by the length of its genomic mRNA. We synthesize viral mRNA of desired lengths using simple molecular biology techniques, and in vitro assemble the mRNA with viral coat proteins to yield viral building blocks of controlled lengths. The results indicate that the assembly of the viral building blocks is consistent and reproducible, and can be readily extended to assemble building blocks with genetically modified coat proteins (TMV1cys). Additionally, we confirm the potential utility of the TMV1cys viral building blocks with controlled dimensions via covalent and quantitative conjugation of fluorescent markers. We envision that our biologically inspired assembly strategy to design and construct viral building blocks of controlled dimensions could be employed to fabricate well-controlled nanoarchitectures and hybrid nanomaterials for a wide variety of applications including nanoelectronics and nanocatalysis.

Selective derivatization of nucleotide diphosphate (NDP)-4-keto sugars for electrospray ionization-mass spectrometry (ESI-MS).

Nucleotide diphosphate (NDP) sugars are widely present in antibiotics and glycoconjugates, such as protein- and lipid-linked oligosaccharides, where they act as substrates for glycosyltransferase in eukaryotes and prokaryotes. Among NDP sugars, NDP-4-keto sugars are key intermediates in the synthesis of structurally diverse NDP sugars with different functional groups. However, the structural identification of the NDP-4-keto sugars via mass spectrometry (electrospray ionization-mass spectrometry (ESI-MS)) continues to be a challenge because of the carbonyl group in these sugars interferes with ionization process. In this study, we evaluated various hydroxylamine compounds for the derivatization of NDP-4-keto sugars, so that the detection of the sugars by ESI-MS is more efficient. As a result, O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine was found to be the most effective tagging molecule for the detection of NDP-4-keto sugars without being interfered by original MS. This method can be used for identifying NDP-4-keto sugars such as thymidine diphosphate (TDP)-, adenosine diphosphate (ADP)-, uridine diphosphate (UDP)-, and cytosine diphosphate (CDP)-4-keto sugars as well as new NDP-4-keto-dehydratases.

Down-regulation of cyclooxygenase-2 and telomerase activity by beta-lapachone in human prostate carcinoma cells.

Beta-lapachone, the product of a tree Tabebuia avellanedae from South America, is known to exhibit various pharmacologic properties, the mechanisms of which are poorly understood. In the present study, we investigated further possible mechanisms by which beta-lapachone exerts its anti-proliferative action in cultured human prostate carcinoma DU145 cells. Exposure of DU145 cells to beta-lapachone resulted in growth inhibition and induction of apoptosis in a dose-dependent manner as measured by MTT assay, fluorescent microscopy, and flow-cytometry analysis. The increase in apoptosis was associated with a dose-dependent up-regulation in pro-apoptotic Bax expression, down-regulation of anti-apoptotic Bcl-2, and proteolytic activation of caspase-3 protease. We found beta-lapachone decreased the levels of cyclooxygenase (COX)-2 mRNA and protein expression without significant changes in the levels of COX-1, which was correlated with a decrease in prostaglandin E2 (PGE2) synthesis. Furthermore, beta-lapachone treatment markedly inhibited the activity of telomerase in a dose-dependent fashion. Additionally, the expression of human telomerase reverse transcriptase (hTERT), a main determinant of the telomerase enzymatic activity, was progressively down-regulated by beta-lapachone treatment. Taken together, these findings provide important new insights into the possible molecular mechanisms of the anti-cancer activity of beta-lapachone.

Induction of apoptosis and inhibition of telomerase activity by aqueous extract from Platycodon grandiflorum in human lung carcinoma cells.

The objective of the present study was to investigate the effect of aqueous extract from the root of Platycodon grandiflorum (AEPG) on the cell growth and apoptosis in human lung carcinoma cell line A549. Exposure of A549 cells to AEPG resulted in growth inhibition and induction of apoptosis in a dose-dependent manner as measured by hemocytometer counts, fluorescence microscopy and flow cytometry analysis. This increase in apoptosis was associated with a decrease in Bcl-2 expression, an increase of Bax and an activation of caspase-3. AEPG treatment markedly inhibited the activity of telomerase in a dose-dependent fashion. Additionally, the expression of human telomerase reverse transcriptase (hTERT), a main determinant of the telomerase enzymatic activity, was progressively down-regulated by AEPG treatment. These findings suggest that the apoptotic events by AEPG were associated with the diminished telomerase activity and down-regulation of Bcl-2 expression.