No abstract

32P labeling of microtubular protein by endogenous protein kinase activity is shown to result from a net increase in protein-bound phosphate and is not the result of a phosphate exchange reaction between ATP and phosphoprotein. Protein phosphorylation is maximal in the presence of 0.5 mM Mg2+ and 0.25 mM ATP, resulting in approximately 2.8 nmol of phosphate/mg of protein. However, phosphorylation can be increased two-to threefold by cAMP. The protein substrates for phosphorylation either the absence or presence of cAMP are the microtubule-associated proteins which copurify with tubulin and promote microtubule assembly. Phosphorylation of microtubule-associated proteins inhibits both the rate and extent of microtubule assembly when the protein is exposed to conditions which result in dissociation of rings. These results are taken to indicate that phosphorylation modifies MAPs so that they have a reduced ability to form an assembly-competent complex with tubulin.

Apoptotic thymocytes were found to be much dimmer than normal thymocytes when stained with several nucleic acid dyes. These dyes provide a quick and simple assay for apoptosis which works for live cells and does not require a UV laser. The collection of dyes giving this staining pattern includes reagents suitable for use in either the FL1, FL2, or FL3 channel of a standard FACScan. Cells identified by these reagents were identical to apoptotic thymocytes defined by several widely used criteria: (i) rapid uptake of Hoechst 33342 but exclusion of propidium iodide, (ii) merocyanin 540 bright, and (iii) sub-GI DNA content when permeabilized in a buffer that elutes fragmented DNA. In addition, L3T4my-l dim thymocytes were included in the dye dim population. The standard Hoechst 33342 and merocyanin 540 assays were not able to separate the normal and apoptotic populations in HL-60 cells treated with camptothecin.However, the dyes mo-16 and LDS-751 both gave adequate differentiation of apoptotic from nonapoptotic cells in this model system. Some of these dyes also emit very little in other fluorescence channels of the flow cytometer and can be used in multicolor assays on cytometers equipped with only a single argon-ion laser. 0 1995 Wiley-Liss, Inc.Key terms: LDS-751, thymocytes, programmed cell death, Apoptosis is a mode of cell death important in a wide variety of situations (5,2054). It is characterized by nuclear chromatin condensation and morphological changes that occur in the absence of plasma membrane breakdown. The nuclear DNA of apoptotic cells is also nicked, although not all model systems show the extensive DNA degradation seen in cells of hematopoietic lineages (4,6,35). Many of the other characteristics of apoptotic cells have been noted in some, but not all, models studied.The differences in the model systems studied has led to the appearance of several different assays for apoptosis (31). Electron microscopic examination is usually considered the most definitive assay. Other less cumbersome assays have been widely used. These include gel electrophoresis to detect the laddering characteristic of internucleosomal cleavage of DNA, flow cytometric detection of cells with "sub-G," DNA content, and various flow cytometric and in situ nick extension assays for the detection of DNA strand breaks (10,17). A potential limitation of all of these assays is that the cells are no longer viable.Some reported assays have been capable of detecting apoptosis in live cells. The DNA dye Hoechst 33342 is more rapidly taken up by apoptotic thymocytes (12,15,18,26). This has been attributed to an increase in membrane permeability. This assay is simple but requires W excitation. An increase in efflux of fluorescein after loading with fluorescein diacetate (26) and an increased influx rate for ethidium bromide (22) have been attributed to the same increased membrane permeability. In a second type of assay, a decrease in cell surface antigens is used to identify apoptotic cells. Antigens studied have included the CD45 antigen ...

Elements from DNA microarray analysis, such as sample labeling and micro-spotting of capture reagents, have been successfully adapted to multiplex measurements of soluble cytokines. Application in cell biology is hampered by the lack of mono-specific antibodies and the fact that many proteins occur in complexes. Here, we incorporated a principle from Western blotting and resolved protein size as an additional parameter. Proteins from different cellular compartments were labeled and separated by size exclusion chromatography into 20 fractions. All were analyzed with replicate antibody arrays. The elution profiles of all antibody targets were compiled to color maps that resemble Western blots with bands of antibody reactivity across the size separation range (670 -10 kDa). A new solid phase designed for processing in microwell plates was developed to handle the large number of samples. Antibodies were bound to protein G-coupled microspheres surface-labeled with 300 combinations of four fluorescent dyes. Fluorescence from particle color codes and the protein label were measured by high-speed flow cytometry. Cytoplasmic protein kinases were detected as bands near predictable elution points. For proteins with atypical elution characteristics or multiple contexts, two or more antibodies were used as internal references of specificity. Membrane proteins eluted near the void volume, and additional bands corresponding to intracellular forms were detected for several targets. Elution profiles of cyclin-dependent kinases (cdks), cyclins, and cyclindependent kinase inhibitors, were compatible with their occurrence in complexes that vary with the cell cycle phase and subcellular localization. A two-dimensional platform circumvents the need for mono-specific capture antibodies and extends the utility of antibody array analysis to studies of protein complexes. Molecular & Cellular Proteomics 8:245-257, 2009.

Myosin Vb (MYO5B) is a motor protein that facilitates protein trafficking and recycling in polarized cells by RAB11- and RAB8-dependent mechanisms. Biallelic MYO5B mutations are identified in the majority of patients with microvillus inclusion disease (MVID). MVID is an intractable diarrhea of infantile onset with characteristic histopathologic findings that requires life-long parenteral nutrition or intestinal transplantation. A large number of such patients eventually develop cholestatic liver disease. Bi-allelic MYO5B mutations are also identified in a subset of patients with predominant early-onset cholestatic liver disease. We present here the compilation of 114 patients with disease-causing MYO5B genotypes, including 44 novel patients as well as 35 novel MYO5B mutations, and an analysis of MYO5B mutations with regard to functional consequences. Our data support the concept that (1) a complete lack of MYO5B protein or early MYO5B truncation causes predominant intestinal disease (MYO5B-MVID), (2) the expression of full-length mutant MYO5B proteins with residual function causes predominant cholestatic liver disease (MYO5B-PFIC), and (3) the expression of mutant MYO5B proteins without residual function causes both intestinal and hepatic disease (MYO5B-MIXED). Genotype-phenotype data are deposited in the existing open MYO5B database in order to improve disease diagnosis, prognosis, and genetic counseling.

Many parallel processes occur during the final stages of apoptosis. It is not clear which of these processes occur in all or most models of apoptosis and which occur only in some. In addition, the temporal relationship of these events is not always well understood. Correlated flow cytometric measurements were used to address these questions. Several models of apoptosis were studied, including thymocytes treated with dexamethasone, MOLT‐4 cells treated with etoposide, U937 cells treated with anti‐Fas, HL‐60 cells treated with camptothecin, Raji cells grown in low serum, and aged neutrophils. All models showed a decrease in LDS‐751 and fluorescein diacetate (FDA) staining, an increase in staining with dihydrorhodamine 123 (dhR123) or dihydroethidium, and an acidification of the cytoplasm. In each model, these changes were highly correlated, appearing simultaneously as multiparameter measurements. Changes in membrane status detected with merocyanin 540 (MC540) and annexin V behaved differently. A population with LDS‐751 and FDA changes but without annexin V or MC540 changes could be demonstrated in some models. Several models did not show any change in annexin V binding, and HL‐60 did not show a change in MC540 binding during apoptosis. The loss of cell surface antigens (CD45 and CD16) from aged neutrophils occurred in the entire LDS‐751 and FDA dim population, even though other membrane changes (including the appearance of annexin V binding sites) were only apparent in a subset of these cells. These results suggest a model for the ordering of some of the terminal processes in apoptosis, with annexin V and MC540 changes trailing other events in apoptosis. These results confirm the need for caution in using a single‐parameter measurement as an indicator of apoptosis for any new model being studied. Cytometry 28:253–263, 1997. © 1997 Wiley‐Liss, Inc.

No abstract

Summary. CD38 expression on chronic lymphocytic leukaemia (CLL) cells is a poor prognostic factor, however, methods for measuring this vary. The QuantiBRITE TM flow cytometry (FC) system yields an absolute antigen expression value (antibodies bound/cell, ABC) and may be useful in standardizing CD38 expression analysis. We evaluated cryopreserved pretreatment CLL cells for CD20 ABC, CD38 ABC, and percentage of CD38 + B cells from 131 patients requiring therapy. The 92 patients (70%) with ‡ 100 CD38 ABC had worse overall survival (OS; 34% at 5 years) compared with those with < 100 CD38 ABC (70% at 5 years, mortality hazard ratio 2AE30, 95% confidence interval 1AE28-4AE12; two-tailed P ¼ 0AE003). Among the 64 patients with < 30% CD38 + cells, OS of the 25 with ‡ 100 ABC was worse than that of the 39 with < 100 ABC (P ¼ 0AE018). OS of patients with < 30% CD38 + cells and ‡ 100 ABC was actually similar to that of patients with ‡ 30% CD38 + cells. Bright CD20 expression ( ‡ 20 000 ABC) was not associated with a worse OS (P ¼ 0AE10). The presence of ‡ 100 CD38 ABC in CLL patients requiring therapy is an unfavourable prognostic factor for OS and quantitative FC may be superior to percentage CD38 + cell assessment. Prospective trials are required to determine more precisely the prognostic significance of absolute expression levels in fresh CLL cells.