Could Egg White Lysozyme be Solved by Single Particle Cryo-EM?

The combination of high-end cryogenic transmission electron microscopes (cryo-EM), direct electron detectors, and advanced image algorithms allows researchers to obtain the 3D structures of much smaller macromolecules than years ago. However, there are still major challenges for the single-particle cryo-EM method to achieve routine structure determinations for macromolecules much smaller than 100 kDa, which are the majority of all plant and animal proteins. These challenges include sample characteristics such as sample heterogeneity, beam damage, ice layer thickness, stability, and quality, as well as hardware limitations such as detector performance, beam, and phase plate quality. Here, single particle data sets were simulated for samples that were ideal in terms of homogeneity, distribution, and stability, but with realistic parameters for ice layer, dose, detector performance, and beam characteristics. Reference data were calculated for human apo-ferritin using identical parameters reported for an experimental data set downloaded from EMPIAR. Processing of the simulated data set resulted in a value of 1.86 Šfrom 20 214 particles, similar to a 2 Šdensity map obtained from 29 224 particles selected from real micrographs. Simulated data sets were then generated for a 14 kDa protein, hen egg white lysozyme (HEWL), with and without an ideal phase plate (PP). Whereas we could not obtain a high-resolution 3D reconstruction of HEWL for the data set without PP, the one with PP resulted in a 2.78 Šresolution density map from 225 751 particles. Our simulator and simulations could help in pushing the size limits of cryo-EM.

Misassembly of full-length Disrupted-in-Schizophrenia 1 protein is linked to altered dopamine homeostasis and behavioral deficits.

Disrupted-in-schizophrenia 1 (DISC1) is a mental illness gene first identified in a Scottish pedigree. So far, DISC1-dependent phenotypes in animal models have been confined to expressing mutant DISC1. Here we investigated how pathology of full-length DISC1 protein could be a major mechanism in sporadic mental illness. We demonstrate that a novel transgenic rat model, modestly overexpressing the full-length DISC1 transgene, showed phenotypes consistent with a significant role of DISC1 misassembly in mental illness. The tgDISC1 rat displayed mainly perinuclear DISC1 aggregates in neurons. Furthermore, the tgDISC1 rat showed a robust signature of behavioral phenotypes that includes amphetamine supersensitivity, hyperexploratory behavior and rotarod deficits, all pointing to changes in dopamine (DA) neurotransmission. To understand the etiology of the behavioral deficits, we undertook a series of molecular studies in the dorsal striatum of tgDISC1 rats. We observed an 80% increase in high-affinity DA D2 receptors, an increased translocation of the dopamine transporter to the plasma membrane and a corresponding increase in DA inflow as observed by cyclic voltammetry. A reciprocal relationship between DISC1 protein assembly and DA homeostasis was corroborated by in vitro studies. Elevated cytosolic dopamine caused an increase in DISC1 multimerization, insolubility and complexing with the dopamine transporter, suggesting a physiological mechanism linking DISC1 assembly and dopamine homeostasis. DISC1 protein pathology and its interaction with dopamine homeostasis is a novel cellular mechanism that is relevant for behavioral control and may have a role in mental illness.

Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.

Mutations of the genes encoding APC or beta-catenin in colon carcinoma induce the constitutive formation of nuclear beta-catenin/Tcf-4 complexes, resulting in activated transcription of Tcf target genes. To study the physiological role of Tcf-4 (which is encoded by the Tcf7/2 gene), we disrupted Tcf7/2 by homologous recombination. Tcf7/2-/- mice die shortly after birth. A single histopathological abnormality was observed. An apparently normal transition of intestinal endoderm into epithelium occurred at approximately embryonic day (E) 14.5. However, no proliferative compartments were maintained in the prospective crypt regions between the villi. As a consequence, the neonatal epithelium was composed entirely of differentiated, non-dividing villus cells. We conclude that the genetic program controlled by Tcf-4 maintains the crypt stem cells of the small intestine. The constitutive activity of Tcf-4 in APC-deficient human epithelial cells may contribute to their malignant transformation by maintaining stem-cell characteristics.

Evaluation of the impact of direct plating, broth enrichment, and specimen source on recovery and diversity of methicillin-resistant Staphylococcus aureus isolates among HIV-infected outpatients.

We compared recovery of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) from nasal and groin swab specimens of 600 HIV-infected outpatients by selective and nonselective direct plating and broth enrichment. Swabs were collected at baseline, 6-month, and 12-month visits and cultured by direct plating to mannitol salt agar (MSA) and CHROMagar MRSA (CM) and overnight broth enrichment with subculture to MSA (broth). MRSA isolates were characterized by pulsed-field gel electrophoresis (PFGE), staphylococcal cassette chromosome mec (SCCmec) typing, and PCR for the Panton-Valentine leukocidin. At each visit, 13 to 15% of patients were colonized with MRSA and 30 to 33% were colonized with methicillin-susceptible S. aureus (MSSA). Broth, CM, and MSA detected 95%, 82%, and 76% of MRSA-positive specimens, respectively. MRSA recovery was significantly higher from broth than CM (P ≤ 0.001) or MSA (P ≤ 0.001); there was no significant difference in recovery between MSA and CM. MSSA recovery also increased significantly when using broth than when using MSA (P ≤ 0.001). Among specimens collected from the groin, broth, CM, and MSA detected 88%, 54%, and 49% of the MRSA-positive isolates, respectively. Broth enrichment had a greater impact on recovery of MRSA from the groin than from the nose compared to both CM (P ≤ 0.001) and MSA (P ≤ 0.001). Overall, 19% of MRSA-colonized patients would have been missed with nasal swab specimen culture only. USA500/Iberian and USA300 were the most common MRSA strains recovered, and USA300 was more likely than other strain types to be recovered from the groin than from the nose (P = 0.05).

Methicillin-resistant Staphylococcus aureus colonization in HIV-infected outpatients is common and detection is enhanced by groin culture.

SUMMARYAlthough high rates of clinical infection with methicillin-resistant Staphylococcus aureus (MRSA) have been reported in HIV-infected adults, data on MRSA colonization are limited. We enrolled HIV-infected adults receiving care at the Atlanta VA Medical Center. Swabs from each participant's nares and groin were cultured with broth enrichment for S. aureus. Of 600 HIV-infected adults, 79 (13%) were colonized with MRSA and 180 (30%) with methicillin-susceptible S. aureus. MRSA pulsed-field gel electrophoresis types USA300 (n=44, 54%) and USA500/Iberian (n=29, 35%) predominated. Inclusion of groin swabs increased MRSA detection by 24% and USA300 detection by 38%. In multivariate analysis, MRSA colonization compared to no MRSA colonization was associated with a history of MRSA clinical infection, rarely or never using condoms, and contact with prisons and jails. In summary, the prevalence of MRSA colonization was high in this study of HIV-infected adults and detection of USA300 was enhanced by groin culture.

Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perforin and granzymes.

Cytotoxic T lymphocytes (CTL) contain granules that are exocytosed during specific interaction with target cells (TC). In this process, the granule contents, including the lethal protein perforin, as well as granzymes, a family of serine esterases, are delivered to the TC. Information regarding the routing of these proteins towards the granule and their exact localization within the granule is of primary importance to resolve the mechanism of granule-mediated TC killing. In this study, the subcellular localization of perforin, granzymes, and known endosomal and lysosomal marker proteins was determined in human and murine CTL, by immunogold labeling of ultrathin cryosections followed by electron microscopy. Perforin and granzymes can be detected in rough endoplasmic reticulum, Golgi complex, trans-Golgi reticulum, and in all cytotoxic granules. Within the granules, they have a similar distribution and are localized not only in the so-called dense core but also over the region containing small internal vesicles. This finding implies that perforin and granzymes can be released in membrane-enveloped and/or -associated form into the intercellular cleft formed upon CTL-TC interaction. On the basis of the present evidence, additional release of these molecules in soluble form cannot be excluded. The lysosomal membrane glycoproteins lamp-1, lamp-2, and CD63, are abundantly present on the granule-delimiting outer membrane, which becomes incorporated into the CTL plasma membrane during lethal hit delivery. In contrast, the cation-dependent mannose 6-phosphate receptor, known to be present in endosomes and absent from lysosomes, is found only in a minority of the granules. Together with our previous findings that the granules are acidic and connected to the endocytic pathway, these observations define CTL granules as secretory lysosomes.

Major histocompatibility complex class II compartments in human B lymphoblastoid cells are distinct from early endosomes.

In human B lymphoblastoid cell lines, the majority of major histocompatibility complex (MHC) class II heterodimers are located on the cell surface and in endocytic compartments, while invariant chain (Ii)-associated class II molecules represent biosynthetic intermediates which are present mostly in the endoplasmic reticulum and Golgi complex. To investigate the origin of the MHC class II-positive compartments and their relation to early endosomes, the intracellular distribution of MHC class II molecules and Ii in relation to endocytic tracers was studied in human lymphoblastoid B cells by immunoelectronmicroscopy on ultrathin cryosections. Cross-linking of surface immunoglobulins, followed by a brief period of internalization of the immune complexes, did not alter the intracellular distribution of MHC class II molecules. While early endosomes were abundantly labeled for the cross-linked immunoglobulins, < 1% of total MHC class II molecules were detectable in early endosomes. MHC class II- and Ii-positive structures associated with the trans-Golgi network can be reached by endocytosed bovine serum albumin (BSA)-gold conjugates after 30 min of internalization. Prolonged exposure to BSA-gold allowed visualization of later endocytic compartments, in which a progressive loss of Ii was observed: first the lumenal portion, and then the cytoplasmic portion of Ii escaped detection, culminating in the formation of MHC class II-positive compartments (MIIC) devoid of Ii. The loss of Ii also correlated with a transition from a multivesicular to a multilaminar, electron-dense MIIC. The intracellular compartments in which class II molecules reside (MIIC) are therefore a heterogeneous set of structures, part of the later aspects of the endocytic pathway.

Peptide-induced stabilization and intracellular localization of empty HLA class I complexes.

The human cell line T2 has been reported to be class I assembly deficient, and accordingly expresses reduced amounts of HLA-A2 and no HLA-B5 at the cell surface. By immunoblotting we observe the steady-state class I heavy chain levels of T2 to be near normal when compared with the identical class I alleles of the wild-type cell line T1. In pulse chase experiments, formation of heavy chain beta 2-microglobulin complexes is observed for both HLA-A2 and HLA-B5. Culture at reduced temperatures (26 or 20 degrees C) does not increase the amount of class I molecules transported, unlike what has been reported for the class I assembly-deficient mouse mutant cell line RMA-S. The HLA-B5 and the HLA-A2 complexes formed by T2 are thermolabile in cell lysates, albeit to different degrees. The thermolability of HLA-B5 can be overcome by addition of HLA-B5-presentable peptides, obtained by trifluoroacetic acid extraction from an HLA-B5-positive cell line, underlining the necessity of peptide for class I stability and indicating that T2-derived class I complexes are devoid of peptide. Cytoplast fusion of T2 cells with RMA-S cells shows the defect in class I assembly of RMA-S to be similar to that of T2. Localization of class I molecules observed by immuno-electron microscopy reveals the accumulation in the T2 cell line of both HLA-B5 and HLA-A2 in the endoplasmic reticulum (ER). Class I molecules are present in all the cisternae of the Golgi complex of T2, but the ratio of HLA-A and -B locus products in the Golgi area differs significantly from that at the cell surface. We conclude that the requirement for peptide in transport of class I molecules manifests itself at a stage beyond the ER, most likely the Golgi area.

Distinct molecular forms of human T cell receptor gamma/delta detected on viable T cells by a monoclonal antibody.

A second type of TCR molecule has been identified on human and murine T lymphocytes, which involves the protein products of the gamma and delta genes. T lymphocytes bearing this receptor may constitute a separate cell lineage with a distinct immune function. We have produced an mAb, which specifically detects human TCR-gamma/delta in native as well as denatured states, this in contrast to previously used anti-gamma chain peptide sera, which only reacted with denatured protein. The receptor occurs in different molecular forms, with or without interchain disulphide bonds, in which a delta chain may or may not be detected by cell surface iodination. The mAb is reactive with all these receptor forms. Therefore, this antibody could be used to determine the expression of TCR-gamma/delta on viable human T lymphocytes. In normal individuals, TCR-gamma/delta was found on a subset composing 2-7% of CD3+ lymphocytes in peripheral blood and 0.1-1.0% in thymus. The majority of these cells do not express the CD4 or CD8 antigens, although a significant percentage of CD8+ cells was found. TCR-gamma/delta+ cells in peripheral blood are resting lymphocytes, as judged by ultrastructural analysis. T cell clones with different receptor types can display MHC-nonrestricted cytolytic activity, which is shown to be induced by the culture conditions, most likely by growth factors such as IL-2. This strongly suggests that TCR-gamma/delta does not play a role in target cell recognition in MHC-nonrestricted cytotoxicity. The anti-TCR-gamma/delta antibody can specifically induce cytotoxic activity in clones expressing the receptor, but in addition inhibit growth factor induced cytotoxicity, which indicates a regulatory role of the TCR-gamma/delta/CD3 complex in MHC-nonrestricted cytotoxicity.

Self-recognition of CD1 by gamma/delta T cells: implications for innate immunity.

The specificity of immunoglobulins and alpha/beta T cell receptors (TCRs) provides a framework for the molecular basis of antigen recognition. Yet, evolution has preserved a separate lineage of gamma/delta antigen receptors that share characteristics of both immunoglobulins and alpha/beta TCRs but whose antigens remain poorly understood. We now show that T cells of the major tissue gamma/delta T cell subset recognize nonpolymorphic CD1c molecules. These T cells proliferated in response to CD1+ presenter cells, lysed CD1c+ targets, and released T helper type 1 (Th1) cytokines. The CD1c-reactive gamma/delta T cells were cytotoxic and used both perforin- and Fas-mediated cytotoxicity. Moreover, they produced granulysin, an important antimicrobial protein. Recognition of CD1c was TCR mediated, as recognition was transferred by transfection of the gamma/delta TCR. Importantly, all CD1c-reactive gamma/delta T cells express V delta 1 TCRs, the TCR expressed by most tissue gamma/delta T cells. Recognition by this tissue pool of gamma/delta T cells provides the human immune system with the capacity to respond rapidly to nonpolymorphic molecules on professional antigen presenting cells (APCs) in the absence of foreign antigens that may activate or eliminate the APCs. The presence of bactericidal granulysin suggests these cells may directly mediate host defense even before foreign antigen-specific T cells have differentiated.

Nevirapine-associated hepatotoxicity was not predicted by CD4 count ≥250 cells/µL among women in Zambia, Thailand and Kenya.

OBJECTIVE: The aim of the study was to determine risk factors for developing severe hepatotoxicity (grade 3 or 4 hepatotoxicity) and rash-associated hepatotoxicity (rash with ≥ grade 2 hepatotoxicity) among women initiating nevirapine-based antiretroviral therapy (ART). METHODS: The Non-Nucleoside Reverse Transcriptase Inhibitor Response Study was a prospective cohort study carried out in Zambia, Thailand and Kenya. Between May 2005 and January 2007, we enrolled antiretroviral-naïve HIV-infected women initiating nevirapine-based ART. At enrollment and at weeks 2, 4, 8, 16 and 24, participants had serum alanine transferase (ALT) and aspartate transaminase (AST) measured and were evaluated clinically for hepatitis and rash. RESULTS: Nevirapine-based ART was initiated in 820 women and baseline ALT or AST results were abnormal (≥ grade 1) in 113 (14%) women. After initiating nevirapine-based ART, severe hepatotoxicity occurred in 41 (5%) women and rash-associated hepatotoxicity occurred in 27 (3%) women. In a multivariate logistic regression model, severe hepatotoxicity and rash-associated hepatotoxicity were both associated with baseline abnormal (≥ grade 1) ALT or AST results, but not with a baseline CD4 cell count ≥250 cells/µL. Three participants (0.4%) died with symptoms suggestive of fatal hepatotoxicity; all three women had baseline CD4 count <100 cells/µL and were receiving anti-tuberculosis therapy. CONCLUSION: Among women taking nevirapine-based ART, severe hepatotoxicity and rash-associated hepatotoxicity were predicted by abnormal baseline ALT or AST results, but not by a CD4 count ≥250 cells/µL. In resource-limited settings where transaminase testing is available, testing should focus on early time-points and on women with abnormal baseline ALT or AST results.

Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence.

Protein localization to the TGN was investigated by examining the subcellular distribution of chimeric proteins in which the cytoplasmic and/or transmembrane domains of the TGN protein, TGN38, were substituted for the analogous domains of the plasma membrane protein, Tac. Using immunofluorescence and immunoelectron microscopy, the COOH-terminal cytoplasmic domain of TGN38 was found to be sufficient for localization of the chimeric proteins to the TGN. Deletion analysis identified an 11-amino acid segment containing the critical sequence, YQRL, as being sufficient for TGN localization. TGN localization was abrogated by mutation of the tyrosine or leucine residues in this sequence to alanine, or of the arginine residue to aspartate. In addition to specifying TGN localization, the 11-amino acid segment was active as an internalization signal, although the property of internalization alone was insufficient to confer TGN localization. Overexpression of chimeric proteins containing TGN localization determinants resulted in their detection at the plasma membrane and in intracellular vesicles, and abolished detection of endogenous TGN38. These results suggest that discrete cytoplasmic determinants can mediate protein localization to the TGN, and reveal a novel role for tyrosine-based motifs in this process.

A lysosomal targeting signal in the cytoplasmic tail of the beta chain directs HLA-DM to MHC class II compartments.

In human B cells, class II molecules of the major histocompatibility complex (MHC-II) accumulate in an endosomal/lysosomal compartment, the MIIC, in which they may encounter and bind peptides. An additional molecule required for MHC-II peptide binding, HLA-DM (DM), has also been localized to the MIIC. Neither the relationship of the MIIC to the endosomal system nor the mechanisms by which DM localizes to the MIIC are understood. To address these issues, DM localization was analyzed in cells that do or do not express MHC-II. DM alpha beta heterodimers were localized in transfected MHC-II-negative HeLa and NRK cells, in the absence of the MHC-II-associated invariant chain, to a prelysosomal/lysosomal compartment by immunofluorescence microscopy. To identify a potential targeting determinant, we analyzed the localization of a chimeric protein, T-T-Mb, in which the cytoplasmic tail of murine DM beta (Mb) was appended to the lumenal and transmembrane domains of a cell surface protein, Tac. Like intact DM, T-T-Mb was localized to a lysosomal compartment in HeLa and NRK cells, as judged by immunofluorescence and immunoelectron microscopy. T-T-Mb was rapidly degraded in this compartment by a process that was blocked by inhibitors of lysosomal proteolysis. The DM beta cytoplasmic tail also mediated internalization of anti-Tac antibody from the cell surface and delivery to lysosomes. Deletion from the DM beta cytoplasmic tail of the tyrosine-based motif, YTPL, resulted in cell surface expression of T-T-Mb and a loss of both degradation and internalization; alanine scanning mutagenesis showed that the Y and L residues were critical for these functions. Similarly, mutation of the same Y residue within full-length DM beta resulted in cell surface expression of DM alpha beta heterodimers. Lastly, T-T-Mb was localized by immunoelectron microscopy to the MIIC in a human B lymphoblastoid cell line. Our results suggest that a motif, YTPL, in the cytoplasmic tail of the beta chain of DM is sufficient for targeting either to lysosomes or to the MIIC.

The cytoplasmic domain mediates localization of furin to the trans-Golgi network en route to the endosomal/lysosomal system.

To investigate the mechanisms of membrane protein localization to the Golgi complex, we have examined the intracellular trafficking of epitope-tagged forms of the mammalian endopeptidase, furin, in stably transformed rat basophilic leukemia cells. Our studies show that furin is predominantly localized to the trans-Golgi network (TGN) at steady state, with smaller amounts present in intracellular vesicles. Biochemical and morphological analyses reveal that furin is progressively delivered to a lysosomal compartment, where it is degraded. Analyses of furin deletion mutants and chimeric proteins show that the cytoplasmic domain is both necessary and sufficient for localization to the TGN in various cell types. Interestingly, deletion of most of the cytoplasmic domain of furin results in a molecule that is predominantly localized to intracellular vesicles, some of which display characteristics of lysosomes. To a lesser extent, the cytoplasmically deleted molecule is also localized to the plasma membrane. These observations suggest the existence of an additional determinant for targeting to the endosomal/lysosomal system within the lumenal and/or transmembrane domains of furin. Thus, the overall pattern of trafficking and steady state localization of furin are determined by targeting information contained within more than one region of the molecule.

ARF6 targets recycling vesicles to the plasma membrane: insights from an ultrastructural investigation.

We have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments. Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system. However, another study on the distribution of ARF6 in subcellular fractions of Chinese hamster ovary (CHO) cells suggested that ARF6 did not localize to endosomes labeled after 10 min of horseradish peroxidase (HRP) uptake, but instead was uniquely localized to the plasma membrane, and that its reported endosomal localization may have been a result of overexpression. Here we demonstrate that at the lowest detectable levels of protein expression by cryoimmunogold electron microscopy, ARF6 localized predominantly to an intracellular compartment at the pericentriolar region of the cell. The ARF6-labeled vesicles were partially accessible to HRP only on prolonged exposure to the endocytic tracer but did not localize to early endocytic structures that labeled with HRP shortly after uptake. Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells. HRP labeling in cells expressing ARF6(Q67L), a GTP-bound mutant of ARF6, was restricted to small peripheral vesicles, whereas the mutant protein was enriched on plasma membrane invaginations. On the other hand, expression of ARF6(T27N), a mutant of ARF6 defective in GDP binding, resulted in an accumulation of perinuclear ARF6-positive vesicles that partially colocalized with HRP on prolonged exposure to the tracer. Taken together, our findings suggest that ARF activation is required for the targeted delivery of ARF6-positive, recycling endosomal vesicles to the plasma membrane.

Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments.

The ARF GTP binding proteins are believed to function as regulators of membrane traffic in the secretory pathway. While the ARF1 protein has been shown in vitro to mediate the membrane interaction of the cytosolic coat proteins coatomer (COP1) and gamma-adaptin with the Golgi complex, the functions of the other ARF proteins have not been defined. Here, we show by transient transfection with epitope-tagged ARFs, that whereas ARF1 is localized to the Golgi complex and can be shown to affect predictably the assembly of COP1 and gamma-adaptin with Golgi membranes in cells, ARF6 is localized to the endosomal/plasma membrane system and has no effect on these Golgi-associated coat proteins. By immuno-electron microscopy, the wild-type ARF6 protein is observed along the plasma membrane and associated with endosomes, and overexpression of ARF6 does not appear to alter the morphology of the peripheral membrane system. In contrast, overexpression of ARF6 mutants predicted either to hydrolyze or bind GTP poorly shifts the distribution of ARF6 and affects the structure of the endocytic pathway. The GTP hydrolysis-defective mutant is localized to the plasma membrane and its overexpression results in a profound induction of extensive plasma membrane vaginations and a depletion of endosomes. Conversely, the GTP binding-defective ARF6 mutant is present exclusively in endosomal structures, and its overexpression results in a massive accumulation of coated endocytic structures.

Cytoplasmic tail-dependent localization of CD1b antigen-presenting molecules to MIICs.

CD1 proteins have been implicated as antigen-presenting molecules for T cell-mediated immune responses, but their intracellular localization and trafficking remain uncharacterized. CD1b, a member of this family that presents microbial lipid antigens of exogenous origin, was found to localize to endocytic compartments that included the same specialized subset of endosomes in which major histocompatibility complex (MHC) class II molecules are proposed to bind endocytosed antigens. Unlike MHC class II molecules, which traffic to antigen-loading endosomal compartments [MHC class II compartments (MIICs)] primarily as a consequence of their association with the invariant chain, localization of CD1b to these compartments was dependent on a tyrosine-based motif in its own cytoplasmic tail.

Novel type of proliferating lymphoplasmacytoid cell with a characteristic spotted immunofluorescence pattern.

We examined bone marrow from myeloma patients for the presence of cells with the characteristics of the clonogenic cell in the myeloma stem cell assay. We identified a novel type of cell that contained cytoplasmic immunoglobulin of the relevant idiotype located in a cytoplasmic spot. This "spotted" Ig could be located in the rough endoplasmic reticulum. Spotted cells are highly proliferative, as evidenced by the nuclear staining with the antibody Ki67, and were found in the bone marrow from most of the myeloma patients studied. This type of cell was also present in patients with immunocytomas, in some cases of benign monoclonal gammopathy, and in patients in the state of polyclonal hypergammaglobulinemia. IgG subclass distribution of so-called spotted cells and plasma cells, found in a patient with pseudo biclonal gammopathy, indicates that spotted cells are intermediate between B cells and plasma cells. Spotted cells express the B cell-associated antigens HB4 and HB6 but do not express other B cluster of differentiation antigens or plasmacytoid antigens tested.

Chemically Induced Cuticle Mutation Affecting Epidermal Conductance to Water Vapor and Disease Susceptibility in Sorghum bicolor (L.) Moench.

Analysis of Sorghum bicolor bloomless (bm) mutants with altered epicuticular wax (EW) structure uncovered a mutation affecting both EW and cuticle deposition. The cuticle of mutant bm-22 was about 60% thinner and approximately one-fifth the weight of the wild-type parent P954035 (WT-P954035) cuticles. Reduced cuticle deposition was associated with increased epidermal conductance to water vapor. The reduction in EW and cuticle deposition increased susceptibility to the fungal pathogen Exserohilum turcicum. Evidence suggests that this recessive mutation occurs at a single locus with pleiotropic effects. The independently occurring gene mutations of bm-2, bm-6, bm-22, and bm-33 are allelic. These chemically induced mutants had essentially identical EW structure, water loss, and cuticle deposition. Furthermore, 138 F2 plants from a bm-22 x WT-P954035 backcross showed no recombination of these traits. This unique mutation in a near-isogenic background provides a useful biological system to examine plant cuticle biosynthesis, physiology, and function.