Kathy M. Ensrud

Distinctive Chromosomal Abnormalities in Histologic Subtypes of Non-Hodgkin's Lymphoma

Using a new high-resolution technique for chromosomal analysis, we have successfully studied biopsy specimens of lymph nodes from 42 of 44 patients with non-Hodgkins lymphoma and have categorized them using the new international histologic formulation and immunologic markers. Abnormalities of the clonal chromosomes were detected in all 42 patients. Three recurrent chromosomal aberrations were found to correlate with certain histologic types: a translocation between chromosomes 18 and 14 in 16 of 19 patients with follicular lymphomas (small cleaved cell, mixed cell, and large cell); a translocation between chromosomes 8 and 14 in 5 of 6 patients with small noncleaved-cell (non-Burkitts) or large-cell immunoblastic lymphoma; and a trisomy 12 in 4 of 11 patients with small-cell lymphocytic lymphoma. Our findings suggest that characteristic chromosomal defects occur in certain lymphoma subtypes and that high-resolution chromosomal analysis promises to become an important tool in improving our basic understanding of lymphoid cancers.

All Patients with Acute Nonlymphocytic Leukemia May Have a Chromosomal Defect

Previous studies of banded marrow chromosomes suggest that half the patients with acute nonlymphocytic leukemia (ANLL) have normal karyotypes. To determine whether high-resolution chromosome analysis could detect additional abnormalities, we studied marrow from 26 patients with ANLL, using methotrexate cell synchronization as well as a direct technique. In 24 patients, including 18 who were untreated, adequate mitoses were obtained. All demonstrated clonal chromosomal abnormalities, which involved a balanced translocation in 11 cases, a complete or partial monosomy in 10, and a trisomy in six. Previously reported recurring defects in ANLL were identified, including t(15;17) in two cases, -7 in two cases, and +8 in three cases. In addition, a new specific abnormality involving band 11q23 was noted in one patient with acute monocytic leukemia and in two with myelomonocytic leukemia. Our results suggest that most, if not all, patients with ANLL have chromosomal changes, and that our new technique may allow more precise identification of subtypes of ANLL with characteristic clinical and hematologic features.

Inhibition of Capacitation-Associated Tyrosine Phosphorylation Signaling in Rat Sperm by Epididymal Protein Crisp-1

Abstract Ejaculated sperm are unable to fertilize an egg until they undergo capacitation. Capacitation results in the acquisition of hyperactivated motility, changes in the properties of the plasma membrane, including changes in proteins and glycoproteins, and acquisition of the ability to undergo the acrosome reaction. In all mammalian species examined, capacitation requires removal of cholesterol from the plasma membrane and the presence of extracellular Ca2+ and HCO3−. We designed experiments to elucidate the conditions required for in vitro capacitation of rat spermatozoa and the effects of Crisp-1, an epididymal secretory protein, on capacitation. Protein tyrosine phosphorylation, a hallmark of capacitation in sperm of other species, occurs during 5 h of in vitro incubation, and this phosphorylation is dependent upon HCO3−, Ca2+, and the removal of cholesterol from the membrane. Crisp-1, which is added to the sperm surface in the epididymis in vivo, is lost during capacitation, and addition of exogenous Crisp-1 to the incubation medium inhibits tyrosine phosphorylation in a dose-dependent manner, thus inhibiting capacitation and ultimately the acrosome reaction. Inhibition of capacitation by Crisp-1 occurs upstream of the production of cAMP by the sperm.

A Comparative Analysis of Expression and Processing of the Rat Epididymal Fluid and Sperm-Bound Forms of Proteins D and E

Abstract The mammalian epididymis secretes numerous proteins important for sperm maturation. Among these are proteins D and E, which belong to the CRISP family (cysteine-rich secretory proteins) and are the product of the Crisp-1 gene. These proteins have been the focus of a number of studies and have been implicated in sperm/egg fusion. Protein D and protein E have been purified to apparent homogeneity in several laboratories. Polyclonal antibodies raised against each protein typically cross-reacted with both proteins, suggesting that they were immunologically similar, if not identical. Our laboratory has previously reported the generation of a monoclonal antibody (mAb 4E9) that recognizes only protein E. Using mAb 4E9, the localization of protein E was shown to be domain specific on the sperm surface and there is processing of the protein in the fluid, with only the lowest molecular weight form associating with sperm. Subsequent purification and amino acid sequencing of protein D confirmed that proteins D and E are nearly identical and differ only by presence of the 4E9 epitope on protein E. Here we report the generation of antibodies to regions of amino acid sequence identity in proteins D and E. Using these antibodies, we demonstrate that protein D associates with the sperm head and that a portion of this protein may be proteolytically processed. In addition, we demonstrate that the proteolytic processing of protein E occurs in the carboxy terminal region of this protein. The data also suggest that a portion of protein D may also undergo processing, similar to that of protein E. Finally, we use these antibodies to demonstrate that proteins D and E are differentially expressed by the epididymal epithelium. Taken together, these data suggest that proteins D and E may have individual roles in sperm function.

The major maturation glycoprotein found on rat cauda epididymal sperm surface is linked to the membrane via phosphatidylinositol

The experiments reported here further characterize a approximately 26[3H] kD cell surface glycoprotein that can be detected on rat cauda epididymal sperm using the galactose oxidase/NaB[3H]4 technique (1). When labeled sperm are treated with PI-PLC the 26[3H] kD is completely released from the cell. The released molecule can be recovered undegraded from incubation supernatant. Release by PI-PLC converts the hydrophobic, membrane-anchored form into a hydrophilic molecule as assessed by partition studies using Triton X114. Isoelectric focusing studies using both untreated (control) and PI-PLC treated samples shows that there is charge heterogeneity with two major peaks at pls of approximately 5.0 and approximately 4.5. We also show for the first time that the molecule persists on ejaculated cells.

The 26 kD protein recognized on rat cauda epididymal sperm by monoclonal antibody 4E9 has internal peptide sequence that is identical to the secreted form of epididymal protein E1

MAb 4E9, raised against a detergent extract of rat cauda epididymal sperm, recognizes a 26 kD glycoprotein that is found on the plasma membrane of the sperm tail in cauda, but not caput, sperm (Moore et al., 1994). It also recognizes an epididymis‐secreted protein that has been shown to be protein E (Xu and Hamilton, 1996). It is felt that the secreted protein becomes associated with sperm, but there has been no biochemical evidence of molecular identity between the secreted and membrane proteins. In this report, the membrane form of the antigen has been purified by reverse phase HPLC. Cyanogen bromide cleavage of the purified protein yielded 3 peptides that were purified, also by reverse phase HPLC. One of the peptides yielded an unambiguous sequence of 34 amino acids that is identical to an internal peptide of the protein found in epididymal fluid. This is the first report showing sequence identity between an epididymis‐secreted protein and a protein of the sperm plasma membrane. Mol. Reprod. Dev. 46:377–382, 1997.

Obtaining antibodies to spindle components.

Publisher Summary This chapter outlines a basic method for the identification of spindle components by raising monoclonal antibodies specific to proteins included in the isolated mitotic spindle. Obtaining antibodies to spindle components involves a series of experimental processes: (a) preparation of immunogens and immunization of animals, (b) cell fusion, (c) screening of hybridoma supernatants, and (d) purification and characterization of antibodies. Although each step is considered labor-intensive and time-consuming, recent progress in hybridoma techniques has made it possible to produce specific antibody probes in a simple and reproducible manner. Preparation of immunogens involves four separate processes: (a) preparation of a homogenous mitotic cell population, (b) stabilization of spindle microtubules, (c) lysis of cells, and (d) separation of the whole stabilized spindle structure from other cellular components. The chapter provides the protocol for preparation of spindles from cultured mammalian [Chinese hamster ovary (CHO)] cells which has to be synchronized at mitosis, and then presents two major protocols used for immunizing mice to increase the chance of success in production of monoclonal antibodies.

Preliminary Observations on a Second Mr∼24,000 Membrane Molecule from Rat Spermatozoaa

Preliminary data are presented to show that the integral membrane glycoprotein found on rat cauda epididymal spermatozoa is one of two Mr = approximately 24,000 molecules that are associated with sperm membranes. The second molecule can be differentiated from the glycoprotein by a variety of antibodies and can be shown to be present in spermatozoa and in residual bodies present in the epididymal fluid. The second molecule can be localized immunocytochemically in spermatids and Leydig cells in the testis.