Mouse model of male germ cell apoptosis in response to a lack of hormonal stimulation

As a prerequisite for studies using mutant mice, we established a mouse model for induction of male germ cell apoptosis after deprivation of gonadotropins and intratesticular testosterone (T). We employed a potent long acting gonadotropin-releasing hormone antagonist (GnRH-A), acyline, alone or in combination with an antiandrogen, flutamide for effective induction of germ cell apoptosis in mice. Combined treatment with continuous release of acyline (3 mg/kg BW/day) with flutamide (in the form of sc pellets of 25 mg) resulted in almost the same level of suppression of spermatogenesis, as judged by testis weight and by germ cell apoptotic index, in 2 weeks as that reported for rats after treatment with 1.25 mg/kg BW Nal-Glu GnRH-A for the same time period. Within the study paradigm, the maximum suppression of spermatogenesis occurred after a single sc injection of high (20 mg/kg BW) dose of acyline with flutamide. The combined treatment resulted in complete absence of elongated spermatids. Germ cell counts at stages VII-VIII showed a significant (P < 0.05) reduction in the number of preleptotene (27.1%) and pachytene spermatocytes (81.9%), and round spermatids (96.6%) in acyline + flutamide group in comparison with controls. In fact, treatment with a single high (20 mg/kg BW) dose of acyline combined with flutamide in mice achieved same or greater level of suppression, measured by germ cell counts at stages VII-VIII, in two weeks when compared with those reported after daily treatment with Nal-Glu GnRH-A for 4 weeks in rats. Both plasma and testicular T levels were markedly suppressed after administration of acyline alone either by miniosmotic pump or by a single sc injection. Addition of flutamide to acyline had no discernible effect on plasma or intratesticular T levels when compared with acyline alone. These results demonstrate that optimum suppression of spermatogenesis through increased germ cell death is only possible in mice if total abolition of androgen action is achieved and further emphasize the usefulness of acyline + flutamide treated mice as a suitable model system to study hormonal regulation of testicular germ cell apoptosis.     

Single base mutation in the type III procollagen gene that converts the codon for glycine 883 to aspartate in a mild variant of Ehlers-Danlos syndrome IV

Experiments were carried out to test the hypothesis that a 19-year-old proband with a mild variant of Ehlers-Danlos syndrome type IV had a mutation in the gene for type III procollagen. cDNA and genomic DNA were analyzed by using the polymerase chain reaction and cloning of the products into M13 filamentous phage. A mutation was found that converted the codon for glycine 883 of the triple-helical domain in one allele for type III procollagen to a codon for aspartate. The polymerase chain reaction introduced a few artifactual single base substitutions. Also, it was difficult to distinguish copies from the two alleles in many of the M13 clones. Therefore, several different strategies and analyses of about 50,000 nucleotide sequences in a series of clones were used to demonstrate that the mutation in the codon for glycine 883 was the only mutation in coding sequences for the triple-helical domain of type III procollagen that could have contributed to the phenotype. The same mutation in the codon for glycine 883 in one allele for type III procollagen was found in the proband's 52-year-old father who also had a mild variant of Ehlers-Danlos syndrome type IV. The type III procollagen synthesized by the proband's fibroblasts was analyzed by polyacrylamide gel electrophoresis. Less type III procollagen was secreted by the proband's fibroblasts than by control fibroblasts. Also, the thermal stability of the type III procollagen synthesized by the proband's fibroblasts was lower than the thermal stability of normal type III procollagen as assayed by brief protease digestion. The results, therefore, demonstrated that the single base mutation that converted the codon of glycine 883 to a codon for aspartate destabilized the entire triple helix of type III procollagen and probably accounted for the mild phenotype of Ehlers-Danlos syndrome type IV seen in the proband and her father.     

Electron crystallography - the waking beauty of structural biology

Since its debut in the mid 1970s, electron crystallography has been a valuable alternative in the structure determination of biological macromolecules. Its reliance on single-layered or double-layered two-dimensionally ordered arrays and the ability to obtain structural information from small and disordered crystals make this approach particularly useful for the study of membrane proteins in a lipid bilayer environment. Despite its unique advantages, technological hurdles have kept electron crystallography from reaching its full potential. Addressing the issues, recent initiatives developed high-throughput pipelines for crystallization and screening. Adding progress in automating data collection, image analysis and phase extension methods, electron crystallography is poised to raise its profile and may lead the way in exploring the structural biology of macromolecular complexes.     

Interactions of two monoclonal antibodies with BNP: high resolution epitope mapping using fluorescence correlation spectroscopy

Structure-function studies of antibody-antigen systems include the identification of amino acid residues in the antigen that interact with an antibody and elucidation of their individual contributions to binding affinity. We used fluorescence correlation spectroscopy (FCS) and alanine-scanning mutagenesis to characterize the interactions of brain natriuretic peptide (BNP) with two monoclonal antibodies. Human BNP is a 32 amino acid residue long cyclic polypeptide with the ring structure confined between cysteines in positions 10 and 26. It is an important cardiovascular hormone and a valuable diagnostic cardiac marker. We compare the binding strength of the N-terminus Alexa488-labeled BNP, native cyclic BNP, BNP alanine-substituted mutants, linear BNP, and its short fragments to determine the individual contributions of amino acid residues included in the continuous antigenic epitopes that are recognized by two different monoclonal antibodies raised toward BNP. Implementation of FCS for these studies offers all of the advantages of solution phase measurements, including high sensitivity, simplicity of manipulation with reagents, and elimination of solid phase interferences or separation steps. Significant differences in the molecular masses of the free and antibody bound BNP results in a substantial ( approximately 2.5-times) increase in the diffusion rates. Determination of the binding constants and inhibition effects by measuring the diffusion rates of the ligand at the single molecule level introduces the ultimate opportunity for researching systems where the fluorescence intensity and/or fluorescence anisotropy do not change upon interaction of the ligand with the protein. Monoclonal antibodies 106.3 and BC203 demonstrate high affinities to BNP and bind two distant epitopes forming robust antibody sandwiches. Both antibodies are used in Abbott diagnostic assays on AxSYM, IMx, and Architect platforms.     

Assessing post-induction cellular response in a recombinant E. coli lactose-inducible system by monitoring β-galactosidase levels

Summary A synthetic lactose-inducible promoter was chosen to study host cell responses to the over-expression of heterologous genes. Fermentations were conducted to compare the effect of induction strategies on the synthesis of -galactosidase versus the production of recombinant protein. The levels of lactose, IPTG and glucose during induction were manipulated to adjust the utilization of lactose as the inducer and/or the carbon source. In addition, the involvement of the gal operon in lactose metabolism was also explored in order to optimize lactose transport and utilization during induction.     

Susceptibilities of Algae and Legionella pneumophila to Cooling Tower Biocides

Nine algal strains and nine Legionella pneumophila strains were tested in laboratory culture for their susceptibility to inhibition by a variety of commercially available microbiocides. The responses ranged from ineffective to effective at 1/100 the manufacturers' recommended pulse doses. Tests were also performed to determine whether the action of the microbiocide was bacteriostatic or bacteriocidal.     

Characterization of and Functional Antigen Presentation by Central Nervous System Mononuclear Cells from Mice Infected with Theiler’s Murine Encephalomyelitis Virus

We examined the phenotype and function of cells infiltrating the central nervous system (CNS) of mice persistently infected with Theiler’s murine encephalomyelitis virus (TMEV) for evidence that viral antigens are presented to T cells within the CNS. Expression of major histocompatibility complex (MHC) class II in the spinal cords of mice infected with TMEV was found predominantly on macrophages in demyelinating lesions. The distribution of I-A^s staining overlapped that of the macrophage marker sialoadhesin in frozen sections and coincided with that of another macrophage/microglial cell marker, F4/80, by flow cytometry. In contrast, astrocytes, identified by staining with glial fibrillary acidic protein, rarely expressed detectable MHC class II, although fibrillary gliosis associated with the CNS damage was clearly seen. The costimulatory molecules B7-1 and B7-2 were expressed on the surface of most MHC class II-positive cells in the CNS, at levels exceeding those found in the spleens of the infected mice. Immunohistochemistry revealed that B7-1 and B7-2 colocalized on large F4/80⁺ macrophages/microglia in the spinal cord lesions. In contrast, CD4⁺ T cells in the lesions expressed mainly B7-2, which was found primarily on blastoid CD4⁺ T cells located toward the periphery of the lesions. Most interestingly, plastic-adherent cells freshly isolated from the spinal cords of TMEV-infected mice were able to process and present TMEV and horse myoglobin to antigen-specific T-cell lines. Furthermore, these cells were able to activate a TMEV epitope-specific T-cell line in the absence of added antigen, providing conclusive evidence for the endogenous processing and presentation of virus epitopes within the CNS of persistently infected SJL/J mice.Theiler’s murine encephalomyelitis virus (TMEV) is a picornavirus that induces a lifelong persistent central nervous system (CNS) infection leading to a chronic CNS demyelinating disease when inoculated intracerebrally into susceptible strains of mice. Infected mice develop progressive symptoms of gait disturbance, spastic hind limb paralysis, and urinary incontinence (39), histologically related to perivascular and parenchymal mononuclear cell infiltration and demyelination of white matter tracts within the spinal cord (8, 9, 38). Several lines of evidence have demonstrated that demyelination is immunologically mediated. These include the ability of nonspecific immunosuppression with cyclophosphamide (37), antithymocyte serum (36), and anti-CD4 or anti-major histocompatibility complex (MHC) class II monoclonal antibodies (MAbs) (14, 16, 63) to inhibit or prevent disease and the ability of TMEV-specific tolerance to prevent induction of disease (28). In the highly susceptible SJL/J mouse strain, current evidence indicates that the myelin damage is initiated by TMEV-specific CD4⁺ T cells targeting virus antigen (16, 28, 45, 46, 54), while the chronic stage of the disease also involves CD4⁺ myelin epitope-specific T cells primed via epitope spreading (48). Thus, the immune response itself may be deleterious to CNS function, as exemplified in humans by multiple sclerosis (MS), for which TMEV infection serves as a model.The identity of the cells responsible for initiating and sustaining immune responses in the CNS remains controversial. The CNS lacks normal lymphatic circulation and tissue and is shielded from the systemic circulation by a specialized continuous vascular endothelium (6). There are specialized cells within the CNS with the potential to present antigens to T cells. In vitro, astrocytes (11, 59) and microglia (3, 13), particularly when treated with gamma interferon (IFN-γ), are capable of expressing MHC class II and presenting antigens to T cells. However, studies such as these have relied on the ability to isolate and continuously culture cells from neonatal or embryonic brain and have assumed that such cells are representative of the adult populations in vivo. Antigen presentation by neonatal cells in long-term culture may not faithfully reproduce the in vivo state in adult animals, as the ability of microglia directly isolated from adult rats to present myelin basic protein (MBP) to T-cell lines in vitro was found to differ from that of neonatally derived microglia (12). In addition, studies using allogeneic bone marrow chimeras between strains of mice or rats have generally supported the idea that cells of hematopoietic origin, i.e., microglia and macrophages, are the principal antigen-presenting cells (APCs) in the CNS active during the initiation of experimental autoimmune encephalomyelitis (EAE) (20, 22, 50). Although they are much more abundant than microglia, astrocytes are less potent when inducing EAE in chimeras (50).The role of antigen presentation in the CNS during TMEV-induced demyelination has not been addressed directly. We previously showed that a relatively large fraction of the CD4⁺, but not CD8⁺, T cells isolated from the spinal cords of TMEV-infected mice expressed high-affinity interleukin-2 (IL-2) receptor (IL-2R), a marker of recent T-cell activation. In addition, TMEV-specific CD4⁺ T cells could be demonstrated in the spinal cord infiltrates of TMEV-infected mice (54). This finding raises the possibility that T cells are locally activated within the target tissue and participate directly in the pathogenesis of disease. Macrophages (5, 41, 56), astrocytes (7, 56), and oligodendroglia (55, 56) in TMEV-infected mice contain virus and conceivably could present viral antigens to pathogenic CD4⁺ T cells within the CNS. Isolated microglia (34) and astrocytes (17) have been shown to support persistent viral infection in vitro, and astrocytes derived from neonatal mice have been shown to present TMEV to T cells in vitro (2). To examine whether CNS cells present viral antigens and participate in the pathogenesis of TMEV-induced demyelination, the expression of MHC class II and B7 costimulatory molecules was examined in detail. Based on our previous results showing that a large proportion of CD4⁺ T cells isolated from the CNS of TMEV-infected mice bear markers of recent activation, we also asked if mononuclear cells isolated from the CNS of TMEV-infected mice were capable of presenting viral antigens leading to the functional activation of Th1 lines in vitro.