Pharmacokinetics and biodistribution of radioimmunoconjugates of anti-CD19 antibody and single-chain Fv for treatment of human B-cell Malignancy

The comparative advantages and disadvantages of intact antibodies and single-chain Fv as immunotoxins and radioimmunoconjugates have been widely discussed but not directly compared. In this study, the in vivo properties of anti-CD19 B43 monoclonal antibody and its derived single-chain Fv (FVS191) were studied in athymic nude mice bearing CD19-positive human lymphomas. B43 mab and FVS191 were labeled with iodine-125 using iodine-beads, and immunoreactivities were determined to be 57% and 72%, respectively. Scatchard analysis showed a similar high affinity for both. The results of pharmacokinetic studies revealed that FVS191 had a rapid biphasic clearance from the circulation (T1/2α = 2.5 min, T1/2β = 3.7 h); The T1/2α and T1/2β phases of B43 mab were determined to be 0.72 h and 57 h respectively. Biodistribution studies compared the uptake of labeled antibodies by CD19-positive and by CD19-negative tumors. The peak percentages of injected dose were 5.7% at 12 h for B43 and 2.45% at 1 h for FVS191. Radiolocalization indices (RI) demonstrated tumor-specific uptake for both, but higher uptake for B43. The optimal RI was seen at 15 min for FVS191 and 6 h for B43. FVS191 was unstable in vivo, approximately 50% of the injected dose being degraded in blood in 100 min. Radioactivity detected in the urine was present mainly as the deiodinized form of FVS191. The results suggest that B43 mab is favored over FVS191 in biodistribution properties and in vivo stability. Because B43 Mab showed early tumor-specific uptake, high RI values, and favorable tissue-to-blood ratios, it is a potential candidate for radioimmunotherapy and immunotoxin therapy of B-cell leukemia and lymphoma. Received: 17 June 1997 / Accepted: 17 June 1998     

Localization of actin filaments in internodal cells of characean algae. A scanning and transmission electron microscope study

New methods of visualizing subcortical actin filament bundles, or fibrils, in Characean internodes confirm that they are associated with chloroplasts at the surface facing the streaming endoplasm, and reveal that they are continuous over long distances. With the scanning electron microscope, an average of four to six fibrils are seen bridging a file of chloroplasts. The same configuration appears in negatively stained preparations of large blocks of chloroplast files connected by actin fibrils. Few branches of the subcortical fibrils are evident. These findings are discussed with respect to the mechanism of cytoplasmic streaming in Characeae.     

Optimization of a glycoengineered Pichia pastoris cultivation process for commercial antibody production

Glycoengineering enabled the production of proteins with human N-linked glycans by Pichia pastoris. This study used a glycoengineered P. pastoris strain which is capable of producing humanized glycoprotein with terminal galactose for monoclonal antibody production. A design of experiments approach was used to optimize the process parameters. Followed by further optimization of the specific methanol feed rate, induction duration, and the initial induction biomass, the resulting process yielded up to 1.6 g/L of monoclonal antibody. This process was also scaled-up to 1,200-L scale, and the process profiles, productivity, and product quality were comparable with 30-L scale. The successful scale-up demonstrated that this glycoengineered P. pastoris fermentation process is a robust and commercially viable process.     

Spatial regulation of Cdc55-PP2A by Zds1/Zds2 controls mitotic entry and mitotic exit in budding yeast

Budding yeast CDC55 encodes a regulatory B subunit of the PP2A (protein phosphatase 2A), which plays important roles in mitotic entry and mitotic exit. The spatial and temporal regulation of PP2A is poorly understood, although recent studies demonstrated that the conserved proteins Zds1 and Zds2 stoichiometrically bind to Cdc55-PP2A and regulate it in a complex manner. Zds1/Zds2 promote Cdc55-PP2A function for mitotic entry, whereas Zds1/Zds2 inhibit Cdc55-PP2A function during mitotic exit. In this paper, we propose that Zds1/Zds2 primarily control Cdc55 localization. Cortical and cytoplasmic localization of Cdc55 requires Zds1/Zds2, and Cdc55 accumulates in the nucleus in the absence of Zds1/Zds2. By genetically manipulating the nucleocytoplasmic distribution of Cdc55, we showed that Cdc55 promotes mitotic entry when in the cytoplasm. On the other hand, nuclear Cdc55 prevents mitotic exit. Our analysis defines the long-sought molecular function for the zillion different screens family proteins and reveals the importance of the regulation of PP2A localization for proper mitotic progression.     

The budding yeast PP2ACdc55 protein phosphatase prevents the onset of anaphase in response to morphogenetic defects

Faithful chromosome transmission requires establishment of sister chromatid cohesion during S phase, followed by its removal at anaphase onset. Sister chromatids are tethered together by cohesin, which is displaced from chromosomes through cleavage of its Mcd1 subunit by the separase protease. Separase is in turn inhibited, up to this moment, by securin. Budding yeast cells respond to morphogenetic defects by a transient arrest in G2 with high securin levels and unseparated chromatids. We show that neither securin elimination nor forced cohesin cleavage is sufficient for anaphase in these conditions, suggesting that other factors contribute to cohesion maintainance in G2. We find that the protein phosphatase PP2A bound to its regulatory subunit Cdc55 plays a key role in this process, uncovering a new function for PP2A(Cdc55) in controlling a noncanonical pathway of chromatid cohesion removal.     

Immunolocalization of two ligninO-methyltransferases in stems of alfalfa (Medicago sativa L.)

Caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) catalyze parallel reactions that are believed to be involved in the biosynthesis of lignin monomers. Antisera specific for alfalfa (Medicago sativa L.) COMT or CCOMT were raised against the enzymes expressed inEscherichia coli, and were used for immunolocalization studies in lignifying alfalfa stem tissue. Both COMT and CCOMT were localized to xylem parenchyma cells, as assessed by light microscopy and immunocytochemistry. Electron microscopy revealed that both enzymes were located in the cytoplasm of xylem parenchyma cells, and to a lesser extent, in the cytoplasm of phloem cells. There was no significant difference in the localization pattern of COMT and CCOMT, suggesting that the two enzymes may be part of a metabolic grid leading to production of lignin monomers in lignifying tissue of mature alfalfa stem internodes.     

The budding yeast Polo-like kinase Cdc5 is released from the nucleus during anaphase for timely mitotic exit

Polo-like kinases are important regulators of multiple mitotic events; however, how Polo-like kinases are spatially and temporally regulated to perform their many tasks is not well understood. Here, we examined the subcellular localization of the budding yeast Polo-like kinase Cdc5 using a functional Cdc5-GFP protein expressed from the endogenous locus. In addition to the well-described localization of Cdc5 at the spindle pole bodies (SPBs) and the bud neck, we found that Cdc5-GFP accumulates in the nucleus in early mitosis but is released to the cytoplasm in late mitosis in a manner dependent on the Cdc14 phosphatase. This Cdc5 release from the nucleus is important for mitotic exit because artificial sequestration of Cdc5 in the nucleus by addition of a strong nuclear localization signal (NLS) resulted in mitotic exit defects. We identified a key cytoplasmic target of Cdc5 as Bfa1, an inhibitor of mitotic exit. Our study revealed a novel layer of Cdc5 regulation and suggests the existence of a possible coordination between Cdc5 and Cdc14 activity.     

CD4+ T-Lymphocyte Depletion in Human Lymphoid Tissue Ex Vivo Is Not Induced by Noninfectious Human Immunodeficiency Virus Type 1 Virions

We tested infectious human immunodeficiency virus type 1 (HIV-1), noninfectious but conformationally authentic inactivated whole HIV-1 virions, and purified gp120 for the ability to induce depletion of CD4⁺ T cells in human lymphoid tissues ex vivo. Infectious CXCR4-tropic HIV-1, but not matched inactivated virions or gp120, mediated CD4⁺ T-cell depletion, consistent with mechanisms requiring productive infection.     

Differentiation in mature T lymphoid leukemia cells is unstable and reversible to myeloid cells, without the involvement of a common stem cell.

Mechanisms for the unstable and reversible differentiation of a mature CD3+ CD7+TCR-alpha/beta+ T lymphoid leukemia into the myeloid lineage were investigated. Inasmuch as productive rearrangement of the TCR-alpha is a late determinant of T cell differentiation, the TCR-alpha rearrangement was sequenced to determine the state of differentiation of the leukemic multipotent cell. An identical productive rearrangement of J alpha C to a novel V alpha region was found in the myeloid and T lymphoid leukemic cells. Thus, a "terminally" differentiated T lymphoid leukemic cell after productively rearranging TCR-alpha and -beta continues to display potential for multilineage differentiation. Therefore, multilineage potential is due to an unstable and reversible differentiation in a mature T lymphocyte as opposed to differentiation of an uncommitted common T and myeloid precursor cell.