Waldenström macroglobulinemia – definition, symptoms, and treatment

Waldenstr?m macroglobulinemia (WM) is a low-grade lymphoplasmacytic lymphoma of mature IgM⁺ B-lymphocytes that remains incurable despite recent practice-altering therapeutic advances and refinements in patient care. Defining features of WM include symptoms that can either be attributed to the extent and site of tissue infiltration by tumor cells or the magnitude and immunological specificity of the monoclonal serum IgM (paraprotein). Current guidelines for the therapeutic stratification of patients with newly diagnosed WM recommend BR (bendamustin-rituximab) for bulky and/or symptomatic disease. DRC (dexamethasone-rituximab-cyclophosphamide) is a good treatment option for relapsed or refractory WM. Ibrutinib – a small-drug inhibitor of Bruton tyrosine kinase, approved for WM treatment in the United States and Europe in 2015 – is particularly effective for tumors that harbor the hallmark MYD88^L265P mutation. Plasma exchange is indicated in patients with IgM-dependent hyperviscosity syndrome. The potential development of novel drugs and combination regimens generates promise that the future of patients with WM is bright.     

Waldenström macroglobulinemia – immunophenotype and natural history

Despite encouraging progress in recent years, our knowledge of the natural history of Waldenstr?m macroglobulinemia (WM), a low-grade LPL (lymphoplasmacytic lymphoma) of mature IgM⁺ B-lymphocytes, remains superficial. This is particularly true of the etiology of WM (tumor causation and initiation) and the sequence of events that underlie the malignant transformation of precursor B cells (tumor progression). Here we briefly review the epidemiology of and genetic predisposition to WM and consider the role of autoimmunity and chronic inflammation in related tumor development. We discuss the immunophenotypic features of WM, including the immunological specificity of WM-associated IgM paraproteins. The proclivity of patients with WM to develop the rare immunoglobulin autoantibody syndromes mixed IgM-IgG cryoglobulinemia, chronic cold agglutinin disease, and IgM neuropathy will also be discussed. We conclude with a call for additional research to elucidate outstanding questions, such as the role of T cell-dependent vs. –independent immune responses in the pathophysiology of WM.     

Experimental model systems for preclinical research on Waldenström macroglobulinemia

Waldenstr?m macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma of mature IgM⁺ B-lymphocytes that warrants additional research to increase therapeutic options, enhance quality of life, and improve survival of patients with WM. Here we concluded a miniseries of short reviews on the diagnosis and treatment ^[1], natural history ^[2] and putative cell-of-origin of WM ^[3] with a brief survey of preclinical experimental model systems available for fundamental and translational research studies on this enigmatic neoplasm. The model systems comprise of: ① continuous tumor cell lines, three of which are well authenticated and demonstrated to be derived from the patient''s index tumor; ② human-in-mouse xenografts that rely on immunodeficient laboratory mice, adapted to carry small fragments of implanted human bone, to provide a suitable microenvironment for incoming lymphoma cells; and ③ genetically engineered mouse models (GEMMs) of neoplastic B-cell development, in which WM-like tumors arise spontaneously in the presence of fully functional innate and adaptive immune systems. Because none of the models developed thus far are perfect, additional efforts are required to achieve a better preclinical representation of disease characteristics of WM. To achieve that goal, the active involvement of basic and clinical research experts from China is called for, so novel drugs and immunotherapies for WM will reach clinics sooner, thereby ensuring the future of patients with WM will be brighter.     

Effect of a mistletoe extract (Iscador® QuFrF) on viability and migratory behavior of human peripheral CD4+ and CD8+ T lymphocytes in three-dimensional collagen lattices

Summary Migration and tissue distribution of immunocompetent cells may be critical prerequisites for efficient immune surveillance. The effect of various concentrations of the mistletoe extract Iscador? QuFrF on the locomotory behavior and viability of immunomagnetically isolated human CD4⁺ and CD8⁺ T lymphocytes within three-dimensional collagen gels was investigated. Although variation in baseline activities of spontaneously migrating T cells was donor-dependent, a dose-dependent stimulation of the locomotory activity in both CD4⁺ and CD8⁺ T cells for noncytotoxic concentrations of Iscador QuFrF (0.25–1.25 μg/ml) was detected. The optimal concentration of mistletoe extract and time of maximal response were specific for each donor. As shown by cell tracking and subsequent data analysis, CD4⁺ T cells exposed to the mistletoe extract displayed a significant increase in mean velocity and time locomoting; total distance migrated was nearly doubled. In contrast, CD8⁺ T cells showed less pronounced changes in these critical parameters. Cytotoxic effects of the mistletoe preparation on T lymphocytes, which could at least partially be attributed to the induction of apoptosis, were drastically reduced in the presence of fetal calf serum in the culture system. Our data suggest that the direct stimulation of T-cell migration in the presence of mistletoe components may modulate in a dose-dependent manner the system of immune surveillance and recognition in patients under mistletoe therapy.     

Epidermal growth factor accelerates recovery of LLC-PK1 cells following oxidant injury

Summary In renal tubular epithelial cells, oxidant injury results in several metabolic alterations including ATP depletion, decreased Na⁺K⁺ ATPase activity, and altered intracellular sodium and potassium content. To investigate the recovery of LLC-PK1 cells following oxidant injury and to determine if recovery can be accelerated, we induced oxidant stress in LLC-PK1 cells with 500 μM hydrogen peroxide for 60 min. Identical cohorts of oxidant-stressed cells were incubated in recovery medium without epidermal growth factor (EGF) or recovery medium containing 25 ng EGF per ml. ATP levels, Na⁺K⁺ ATPase activity in whole cells, Na⁺K⁺ ATPase activity in disrupted cells, and intracellular sodium and potassium ion content were determined at 0, 5, 24, 48, and 72 h following oxidant injury in each cohort of cells. In oxidant-stressed cells recovering in medium without EGF, ATP levels, Na⁺K⁺ ATPase activity, and intracellular ion content improved but continued to remain substantially lower than control values at all time points following oxidant stress. In cells recovering in medium with EGF, ATP levels, Na⁺K⁺ ATPase activity, and the intracellular potassium-to-sodium ratio were significantly higher at nearly all time points than values in cells recovering in medium alone. In cells recovering with added EGF, Na⁺K⁺ ATPase activity had improved to control levels, whereas ATP levels and intracellular ion content approached control values by 72 h following oxidant stress. We conclude that oxidant-mediated ATP depletion, altered Na⁺K⁺ ATPase activity, and intracellular ion content remain depressed for several d following oxidant stress and that EGF accelerated recovery of LLC-PK1 cells from oxidant injury.     

Biological effects of ion beams in Nicotiana tabacum L.

The biological effects of ion beams on Nicotiana tabacum L., particularly the induction of chromosome aberrations, were investigated. Dry seeds were exposed to ¹²C⁵⁺, ⁴He²⁺ and ¹H⁺ beams with linear energy transfer (LET) ranging from 1 to 111 keV/μm and irradiated with gamma-rays. Ion beams were more effective in reducing germination and survival of the seeds than gamma-rays. The LD₅₀ for ¹²C⁵⁺ beams, ⁴He²⁺ beams and gamma-rays were 35, 60 and 500 Gy, respectively. The frequencies of mitotic cells with chromosome aberrations, such as chromosome bridges, acentric fragments and lagging chromosomes in the root tip cells of the exposed seeds, increased linearly with increasing doses. Relative biological effectiveness (RBE) values, based on the doses that induced a survival inhibition of 50% and a 10% frequency of aberrant cells, were 14.3–17.5 for the ¹²C⁵⁺ beams, 7.0–8.3 for the ⁴He²⁺ beams and 7.8 for the ¹H⁺ beams. Furthermore, the relative ratios of the chromosome aberration types were significantly different between the ion beam and the gamma-ray regimes: chromosome fragments were more frequent in the former, and chromosome bridges in the latter. Based on these results, we concluded that the repair process of initial lesions induced by ion beams may be different from that induced by low- LET radiation. Received: 29 October 1998 / Accepted in revised form: 25 March 1999     

In vitro analysis of venom from the wasp Nasonia vitripennis: Susceptibility of different cell lines and venom-induced changes in plasma membrane permeability

Summary The lethal effects of crude venom prepared from the ectoparasitic wasp Nasonia vitripennis were examined with cultured cells from six insect and two vertebrate species. Venom caused cells from Sarcophaga peregrina (NIH SaPe4), Drosophila melanogaster (CRL 1963), Trichoplusia ni (TN-368 and BTI-TN-5B1-4), Spodoptera frugiperda (SF-21AE), and Lymantria dispar (IPL-Ldfbc1) to round up, swell, and eventually die. Despite similar sensitivities and overlapping LC₅₀ values [0.0004–0.0015 venom reservoir equivalents (VRE)/μl], profound differences were noted at the onset of cytotoxicity among the six insect cell lines: over 80% of the NIH SaPe4 and SF21AE cells were nonviable within 1 h after addition of an LC₉₉ dose of venom, whereas the other cells required a 5–10-fold longer incubation period to produce mortality approaching 100%. In contrast, cells from the grass frog, Rana pipiens (ICR-2A), and goldfish, Carassius auratus (CAR), showed little sensitivity to the venom: six venom reservoir equivalents were needed to induce 50% mortality in ICR-2A cells [50% lethal concentration (LC₅₀)=0.067 VRE/μl), and 9 VRE did not yield sufficient mortality in CAR cells for us to calculate an LC₅₀. All susceptible cells showed similar responses when incubated with wasp venom: retraction of cytoplasmic extensions (when present), blebbing of the plasma membrane, swelling of the plasma and nuclear membranes, condensation of nuclear material, and eventual cell death attributed to lysis. The rate of swelling and lysis in NIH SaPe4 and BTI-TN-5B1-4 cells exposed to venom appeared to be dependent on the diffusion potential of extracellular solutes (Na⁺=choline>sucrose≥raffinose>K⁺), which is consistent with a colloid-osmotic lysis mechanism of cell death. When T. ni cells were cotreated with venom and the K⁺ channel blocker 4-aminopyridine, cell swelling and lysis increased with increasing drug concentration. In contrast, cells from S. peregrina were protected from the effects of the venom when treated in a similar manner. Addition of certain divalent cations (Zn⁺² and Ca⁺²) to the extracellular media 1 h postvenom incubation rescued both BTI-TN-5B1-4 and NIH SaPe4 cells, suggesting that protection was gained from closure of open pores rather than prevention of pore formation. Venom from N. vitripennis displayed no hemolytic activity toward sheep erythrocytes, supporting the view that venom intoxication is not by a nondiscriminate mechanism. A possible mode of action of the venom is discussed.     

Expression of Pdx-1 in bone marrow mesenchymal stem cells promotes differentiation of islet-like cells in vitro

Bone marrow mesenchymal stem cells (BMSCs) have the ability of self-renewal and multi-directional differentiation. Recent reports showed that BMSCs could differentiate into endocrine cells of pancreas. However, the differentiation is not efficient enough to produce insulin-producing cells for the future therapeutic use. Pdx-1 is a crucial regulator for pancreatic development. Therefore we constructed a eukaryotic expression vector containing Pdx-1 to determine the effect of Pdx-1 expression on differentiation of BMSCs in vitro. The results showed that BMSCs could self-assemble to form functional pancreatic islet-like structures after differentiation in vitro. The proportion of insulin-producing cells differentiated from Pdx-1⁺BMSCs was 28.23%±2.56%, higher than that from BMSCs transfected with vacant vector and Pdx-1 ⁻ BMSCs (7.23%±1.56% and 4.08%±2.69% respectively) by flow cytometry. Immunocytochemical examination also testified the expression of multiple β-cells-specific genes such as insulin, glucagons, somatostatin in differentiated BMSCs. The results also revealed that the expressions of genes mentioned above in Pdx-1⁺BMSCs were higher than that in Pdx-1⁻BMSCs, which was confirmed by Western blotting analysis and RT-PCR. Glucose-induced insulin secretion from Pdx-1⁺BMSCs in 5mmol/L and 25mmol/L glocuse was (56.61±4.82) μU/mL and (115.29±2.56) μU/mL respectively, which were much higher than those from Pdx-1⁻BMSCs((25.53±6.49) μU/mL and (53.26±7.56) μU/mL respectively). Grafted animals were able to maintain their body weight and survive for relatively longer periods of time than hyperglycemic sham-grafted controls, which demonstrated an overall beneficial effect of the grafted cells on the health of the animals. These findings thus suggested that exogenous expression of Pdx-1 should provide a promising approach for efficiently producing islet-like cells from BMSCs for the future therapeutic use in diabetic patients.     

Differentiation of immortalized epithelial cells derived from cystic fibrosis airway submucosal glands

Summary Cystic fibrosis (CF) involves abnormalities in mucus production and secretion of the airway. Studies of the regulation of airway mucin production and secretion has been difficult due to the lack of in vitro models of the airway epithelial cells which express functional differentiation. Because the majority of the mucin in the airway is apparently produced by the submucosal glands, we have focused our attention on the development of cell culture models of human airway submucosal glands. This report describes the propagation of CF airway submucosal gland epithelial cells which continue to express mucin production. The CF bronchus was obtained from a 31-yr-old patient who received a double lung transplant. The glands were dissected out and primary cultures prepared by the explant/outgrowth procedure. The cells were immortalized by infection with Adl2-SV40 hybrid virus. The cultures are maintained in serum-free keratinocyte basal medium supplemented with insulin (5μg/ml), hydrocortisone (0.5μg/ml), epidermal growth factor (10 ng/ml), bovine pituitary extract (25μg/ml), and antibiotics. Cultures were passaged using 0.125% trypsin in Ca⁺² and Mg⁺²-free Hanks’, balanced salt solution. Polymerase chain reaction (PCR) analysis demonstrated that the cells were homozygous for the ΔF508 mutation. Morphologic observations showed that the cells were epithelial and were interconnected by sparsely distributed desmosomes. Their cytoplasm contained secretory-type structures including abundant Golgi, rough endoplasmic reticulum, and secretory vesicles. Immunofluorescent studies determined that all cells were positive for cytokeratins, mucin glycoconjugates, and cystic fibrosis transmembrane conductance regulator. The cultures secreted substantial amounts of mucin glycoproteins and expressed the MUC-2 mucin gene. Patch clamp experiments revealed that the cells expressed defective Cl⁻ channels which were not activated by Forskolin.     

Massive culture of human liver cancer cells in a newly developed radial flow bioreactor system: Ultrafine structure of functionally enhanced hepatocarcinoma cell lines

Summary With a view to initiating clinical trials, cell morphology and function for a newly developed artificial liver support system employing highly functional human liver cell line, FLC-7, cultured in a radial flow bioreactor were compared to cells grown in a conventional monolayer culture. The radial flow bioreactor consists of a vertically extended cylindrical matrix comprised of porous glass bead microcarriers through which liquid medium flows from the periphery in toward the central axis generating a beneficial concentration gradient of oxygen and nutrients, while preventing excessive shear stresses or buildup of waste products. The three-dimensional culture system supports high-density (1.1 × 10⁸ cells/ml-matrix), large scale cultures (4.4 × 10¹⁰ cells/400 ml-bioreactor) with long-term viability. Scanning and transmission electron microscopy (SEM and TEM) revealed that cells cultured in a monolayer system were flattened and extended with numerous cytoplasmic projections. Cells in the three-dimensional culture were spherical and covered with microvillilike processes resembling liver cells in vivo. The cells were solidly attached on the surfaces and within the pores of the microcarriers in highly dense colonies. The spherical cells remained in close contact with adjacent cells, while circulation of liquid medium flowed freely through spaces between cells. FLC-7 cells produced albumin at a rate of 6.41 μg/24 h/10⁶ cells. Alpha-fetoprotein (AFP) production dropped nearly threefold in comparison to monolayer cultures. Results demonstrated that the new artificial liver support systems (ALSS) provides a superior three-dimensional culture environment that allows cells to perform at naturally functioning levels.     

Nuclear pore dynamics during pollen development and androgenesis in Brassica napus

Changes in nuclear pore complex (NPC) densities, NPCs/nucleus and NPCs/μm³, are described using freeze-fractured Brassica napus microspores and pollen in vivo and in vitro. Early stages of microspore- and pollen-derived embryogenic cells were also analysed. The results of in vivo and in vitro pollen development indicate an increase in activity of the vegetative nucleus during maturation of the pollen. At the onset of microspore and pollen culture, NPC density decreased from 15 NPCs/μm² at the stage of isolation to 9 NPCs/μm², under both embryogenic and non-embryogenic conditions. This implies that the drop in NPC density might be a result of culturing the microspores and pollen rather than an indication for microspore and pollen embryogenesis in Brassica napus. However, after 1 day in culture under embryogenic conditions, the NPC density increased again and stabilised around 13 NPCs/μm², whereas under non-embryogenic conditions the NPC density remained about 9 NPCs/μm². This low density of 9 NPCs/μm² was also found in the nuclei of sperm cells, in contrast to the 19 NPCs/μm² found in the vegetative nucleus. It means that, although both the vegetative and sperm nuclei are believed to be metabolically rather inactive in mature pollen, the NPC density of vegetative nucleus is twice as high as the NPC density of the sperm nuclei. In a few cases, embryos formed suspensor-like structures with a NPC density of 9 NPCs/μm², indicating a lower nucleocytoplasmic exchange of the nuclei of the suspensor cells than with the nuclei in the embryo proper. In addition, observations on NPCs and other organelles, obtained by high resolution cryo-scanning microscopy, are presented. Received: 29 December 1999 / Revision accepted: 3 March 2000     

Soil salt and nutrient concentration in the rhizosphere of desert halophytes

North-West China is an arid region where halophyte plants are rich. Very little is known on the rhizospheric soil of the halophytes in this arid desert region. We conducted a rhizobag experiment on the desert Solonchak soil to investigate the salt and nutrient content in the rhizospheric soil of the desert halophytes. The total salt and the concentrations of 8 major kinds of salt ions increased in the rhizosphere of both succulent halophytes and salt secreting halophytes, but this increase was insignificant for salt-resisting halophytes. Accumulation of Cl^– and Na⁺ is the most significant among the 8 major kinds of salt ions. Accumulation of Cl^– was more significant than that of SO₄^2– in succulent halophytes and salt secreting halophytes. The Na⁺/K⁺, Na⁺/Ca²⁺ and Na⁺/Mg²⁺ ratios in the rhizosphere of all 7 kinds of halophytes were higher than those in the bulk soil. Total N increased significantly in the rhizosphere, but total P and total K decreased. However, the available N, P and K in the rhizosphere of the 7 kinds of halophytes except Phragmites communis Trin. behaved in such an opposite way that available N decreased but available P and available K increased. The ionic contents in the aboveground parts were higher than those in the underground parts of the 7 kinds of halophytes, in particular of both the succulent halophytes and the salt secreting halophytes. Accumulation of Cl^– and Na⁺ in the aboveground parts of the plants was the most significant among that of the 8 major kinds of salt ions.     

Effect of selective and non-selective cysteine protease inhibitors on the intracellular processing of interleukin 6 by HepG2 cells

Summary The effects were measured and compared of three nonselective cysteine cathepsin inhibitors (leupeptin, trans-Epoxysuccinyl-l-Leucylamido(4-guanidino)-butane (E-64), and Z-Phe-Ala-CH₂F) and a selective cathepsin B inhibitor, CA074Me, on the intracellular processing of ¹²⁵I-labeled human recombinant Interleukin 6 (IL-6) by HepG2 cells. The uptake and processing of ¹²⁵I-IL-6 by cells treated with inhibitors was followed over a 7-h period. All inhibitors caused an increased residence time of IL-6 inside the cell and a corresponding decrease in the output of non-trichloroacetic acid-precipitable fragments of radiolabeled protein. Maximal effect was achieved with leupeptin at 200 μM, with which the rate of IL-6 digestion was reduced to 50% that of control cells. The specific inhibitor CA074Me was the least effective in slowing the intracellular processing of IL-6. The effects of all of the inhibitors on the production of haptoglobin, either stimulated by IL-6 or basal, was negligible over a similar time period, indicating continued cell viability. The data from this model suggest that cathepsin inhibitors would not interfere with lysosomal processing to an extent which would prohibit the development of selective and potent cathepsin inhibitors for the treatment of diseases in which individual cysteine cathepsins play clearly pathophysiological roles.     

Effects of mono- and multi-valent cations on the inward-rectifying potassium channel in isolated protoplasts from maize roots

Transport properties mediated by ionic channels were studied by the patch-clamp technique in protoplasts from cortical parenchyma cells of maize roots (CPMR). While outward currents could be seen only occasionally, macroscopic voltage- and time-dependent potassium-selective inward currents (I_K+in) were frequently observed in the whole-cell configuration. These currents increased continuously as a function of K⁺ concentration (in the range 3 – 200 mm) and the slow-saturating macroscopic chord-conductance was fitted by a Michaelis-Menten function with K_m = 195 ± 39 mm. Other ions, like sodium and lithium, did not permeate at all through the maize root inward-channel, or like ammonium (P_NH4+/ P_K+ = 0.16 0.25) and rubidium (P_Rb+/P_K+≈ 0.10) displayed a very low permeability ratio. Up to 5 mm Rb⁺ did not induce any inhibition of the K⁺ inward current, whereas submillimolar concentrations of Cs⁺ were sufficient to block, in a voltage-dependent manner, the inward currents. A decrease of the external potassium concentration favoured Cs⁺ inhibition (K_m = 89 ± 6 μm and 26 ± 2 μm in 200 and 100 mm KCl, respectively). The potassium inward-currents were reversibly and consistently inhibited by submillimolar external concentrations of the metal ions Ni²⁺, Zn²⁺ and Co²⁺, while 1 mm La³⁺ only slightly decreased (≈10%) both the single channel conductance (9.2 ± 1.2 pS in 100 mm potassium) and the macroscopic current. In contrast to the case with Cs⁺, inhibition induced by other metal ions did not show any voltage dependence. These results suggest that, as with animal potassium channels, the inward channel of maize-root cortical cells has a narrow pore of permeation and metal ions decrease the K⁺ current, possibly by acting on binding sites located outside the pore. Received: 21 February 1997 / Accepted: 27 May 1997     

Geometric control of switching between growth, apoptosis, and differentiation during angiogenesis using micropatterned substrates

Summary Past studies using micropatterned substrates coated with adhesive islands of extracellular matrix revealed that capillary endothelial cells can be geometrically switched between growth and apoptosis. Endothelial cells cultured on single islands larger than 1500 μm² spread and progressed through the cell cycle, whereas cells restricted to areas less than 500 μm² failed to extend and underwent apoptosis. The present study addressed whether island geometries that constrained cell spreading to intermediate degrees, neither supporting cell growth nor inducing apoptosis, cause cells to differentiate. Endothelial cells cultured on substrates micropatterned with 10-μm-wide lines of fibronectin formed extensive cell-cell contacts and spread to approximately 1000 μm². Within 72 h, cells shut off both growth and apoptosis programs and underwent differentiation, resulting in the formation of capillary tube-like structures containing a central lumen. Accumulation of extracellular matrix tendrils containing fibronectin and laminin beneath cells and reorganization of platelet endothelial cell adhesion molecule-positive cell-cell junctions along the lengths of the tubes preceded the formation of these structures. Cells cultured on wider (30-μm) lines also formed cell-cell contacts and aligned their actin cytoskeleton, but these cells spread to larger areas (2200 μm²), proliferated, and did not form tubes. Use of micropatterned substrates revealed that altering the geometry of cell spreading can switch endothelial cells among the three major genetic programs that govern angiogenesis—growth, apoptosis and differentiation. The system presented here provides a well-defined adhesive environment in which to further investigate the steps involved in angiogenesis.     

Localization and Quantification of Plasma Membrane Aquaporin Expression in Maize Primary Root: A Clue to Understanding their Role as Cellular Plumbers

Water movement across root tissues occurs by parallel apoplastic, symplastic, and transcellular pathways that the plant can control to a certain extent. Because water channels or aquaporins (AQPs) play an important role in regulating water flow, studies on AQP mRNA and protein expression in different root tissues are essential. Here, we quantified and localized the expression of Zea mays plasma membrane AQPs (ZmPIPs) in primary root tip using in situ and quantitative RT-PCR and immunodetection approaches. All ZmPIP genes except ZmPIP2;7 were expressed in primary roots. Expression was found to be dependent on the developmental stage of the root, with, in general, an increase in expression towards the elongation and mature zones. Two genes, ZmPIP1;5 and ZmPIP2;5, showed the greatest increase in expression (up to 11- and 17-fold, respectively) in the mature zone, where they accounted for 50% of the total expressed ZmPIPs. The immunocytochemical localization of ZmPIP2;1 and ZmPIP2;5 in the exodermis and endodermis indicated that they are involved in root radial water movement. In addition, we detected a polar localization of ZmPIP2;5 to the external periclinal side of epidermal cells in root apices, suggesting an important role in water uptake from the root surface. Finally, protoplast swelling assays showed that root cells display a variable, but globally low, osmotic water permeability coefficient (P _f < 10 μm/s). However, the presence of a population of cells with a higher P _f (up to 26 μm/s) in mature zone of the root might be correlated with the increased expression of several ZmPIP genes.     

Site-specific biotinylation of human myeloid differentiation protein 88 in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> Schneider 2 cell cytoplasm

In this work we describe the production of site-specific biotinylated human myeloid differentiation factor 88 (MyD88). A vector containing a coding sequence for a peptide derived from the carboxyl terminus of the Klebsiella pneumoniae oxalacetate decarboxylase α subunit was used to allow expression and biotinylation of MyD88 in Drosophila melanogaster Schneider 2 cell cytoplasm. As estimated by a comparison of Schneider 2 lysate with standard protein, the maximum expression level was 1.3 μg 10⁷ cells⁻¹. About 4 mg of biotinylated protein was purified by affinity chromatography on monomeric avidin from a I-L culture. Exogenous biotin added to the culture medium increased the biotinylation efficiency of the expressed protein. Biotinylated MyD88 produced in Drosophila cells was able to precipitate recombinant MyD88 expressed in human embryonic kidney cells. The stable expression of MyD88 in Drosophila Schneider 2 cells offers a convenient and attractive method for large-scale production, which may be required to clarify the role of MyD88 in the inflammatory response. Moreover, site-specific biotinylation of MyD88 provides a useful tag for interaction assays where high sensitivity is required.     

Root morphology and cadmium uptake kinetics of the cadmium-sensitive rice mutant

To understand the physiological mechanism that confers Cd sensitivity, root morphology and Cd uptake kinetics of the Cd-sensitive mutant and wild type rice were investigated. The root length, root surface area, and root number of mutant rice decreased more significantly with increasing Cd concentration in growth media compared with the wild type rice. The uptake kinetics for ¹⁰⁹Cd²⁺ in roots of both the mutant and wild type rice were characterized by a rapid linear phase during the first 6 h and a slower linear phase during the subsequent period. Concentration-dependent Cd²⁺ influx in both species could be characterized by the Michaelis-Menten equation, with similar apparent K_m values for mutant and wild type rice (2.54 and 2.37 μM, respectively). However, the V_max for Cd²⁺ influx in mutant root cells was nearly 2-fold higher than that for wild type rice, indicating that enhanced absorption into the root is one of the mechanisms involved in Cd sensitivity in mutant rice.     

中华鳞毛蕨配子体发育过程的观察

Morphological variation during the gametophyte development process of Dryopteris chinensis ( Bak.) Koidz. was observed. The results show that the development process of D. chinensis can be divided into spore germination, filament formation, plate formation, prothallus formation, sexual organ formation and apogamety. The spores are bilaterally symmetric, monolete, surface with ridge fold ornamentation, elliptical in polar view, and approximately semicircle-shaped in equatorial view. The spore germination type is of Vittaria-type;the filaments with a length of 3-7 cells, not branched or occasionally branched, with single or double row cells; mature prothallus is symmetrically cordate, and the development type of prothallus is of Aspidium-type, with long unicellular clavate trichomes distributed on the surface and in the margin;with antheridium but archegonium is not observed, belongs to apogamety. One prothallium of D. chinensis produces one embryo, and young embryo can be generated within one month after the formation of antheridium; there are a lot of unicellular trichomes and some multicellular trichomes on the young embryo of sporophyte.     

Modulation of response to adenosine in vascular smooth muscle cells cultured in defined medium

Summary Cultured pig aortic smooth muscle cells maintain a viable, quiescent state in a chemically defined medium that contains 10⁻⁶ M insulin, 5μg/ml transferrin, and 0.2 mM ascorbate. DNA synthesis and DNA content were determined by measuring tritiated thymidine incorporation and DNA-binding to the fluorescent probe 4′,6-diamidino-2-phenylindole, respectively. The majority of the population of cells in defined medium cultures were diploid. Tritiated thymidine uptake in cells in defined medium was one-tenth that observed in cells in fetal bovine serum-containing medium. The study of cellular cyclic AMP level in response to extracellular adenosine stimulation in dividing cells and quiescent cells showed that cells in defined medium had a lower extent of response to adenosine compared to cells cultured in serum-containing medium. Both the cell growth index and the response to adenosine of cells cultured in defined medium were reversible after replacing the medium with 10% fetal bovine serum-containing medium, which suggests that the cells in defined medium were healthy and were capable of modulating cellular metabolism depending on culture conditions. This work was supported in part by National Institutes of Health grants HL31854, HL38130, and RR07048.