👤 Andrew A Dym

The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (approximately 220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens. Show less

SPINE (Structural Proteomics In Europe) was established in 2002 as an integrated research project to develop new methods and technologies for high-throughput structural biology. Development areas were broken down into workpackages and this article gives an overview of ongoing activity in the bioinformatics workpackage. Developments cover target selection, target registration, wet and dry laboratory data management and structure annotation as they pertain to high-throughput studies. Some individual projects and developments are discussed in detail, while those that are covered elsewhere in this issue are treated more briefly. In particular, this overview focuses on the infrastructure of the software that allows the experimentalist to move projects through different areas that are crucial to high-throughput studies, leading to the collation of large data sets which are managed and eventually archived and/or deposited. Show less

This paper reviews the developments in high-throughput and nanolitre-scale protein crystallography technologies within the remit of workpackage 4 of the Structural Proteomics In Europe (SPINE) project since the project's inception in October 2002. By surveying the uptake, use and experience of new technologies by SPINE partners across Europe, a picture emerges of highly successful adoption of novel working methods revolutionizing this area of structural biology. Finally, a forward view is taken of how crystallization methodologies may develop in the future. Show less

Male infertility is now a major reproductive health problem because of an increasing number of environmental pollutants and chemicals, which eventually result in gene mutations. Genetic alterations caused by environmental factors account for a significant percentage of male infertility. Microarray technology is a powerful tool capable of measuring simultaneously the expression of thousands of genes expressed in a single sample. Eventually, advances in genetic technology will allow for the diagnosis of patients with male infertility due to congenital reasons or environmental factors. Since its introduction in 1994, microarray technology has made significant advances in the identification and characterization of novel or known genes possibly correlated with male infertility in mice, as well as in humans. This provides a rational basis for the application of microarray to establishing molecular signatures for the diagnosis and gene therapy targets of male infertility. In this review, the differential gene expression patterns characterized by microarray in germ and somatic cells at different steps of development or in response to stimuli, as well as a number of novel or known genes identified to be associated with male infertility in mice and humans, are addressed. Moreover, issues pertaining to measurement reproducibility are highlighted for the application of microarray data to male infertility. Show less

Failure to treat deep vein thrombosis (DVT) is associated with significant morbidity and mortality. Anticoagulation, although effective at preventing clot progression, is not able to prevent postthrombotic syndrome. Catheter-directed thrombolysis is a more aggressive alternative, with some small studies suggesting a better long-term outcome, but the associated risks are significant, and the treatment can require 2-3 days in a monitored setting. This report describes the power pulse technique, in which mechanical thrombectomy is combined with thrombolytic agents to maximize the effectiveness of the treatment and reduce the need for prolonged infusion and its associated risks. A 24-patient retrospective study showed complete thrombus removal (>90%) in 12 patients, substantial thrombus removal (50%-90%) in seven patients, and partial thrombus removal (<50%) in five patients. All 24 patients had resolution of presenting symptoms. Only two patients required blood transfusion, and one patient experienced temporary nephropathy. Show less

The feeding response following administration of the free fatty acid oxidation inhibitor, mercaptoacetate (MA) is conceptualized as an experimental model of lipoprivation, which may contribute to the understanding of inter-individual differences in the modulation of this homeostatic response. Although variation in the intake of food, water and glucoprivation as well as intake of several nutrients is known to be associated with genetic variation, it is not known whether MA-induced feeding is similarly dependent upon genotype. The present study therefore examined MA-induced feeding in mice of 11 inbred (A/J, AKR/J, BALB/cJ, CBA/J, C3H/HeJ, C57BL6/J, C57BL10/J, DBA/2J, SJL/J, SWR/J, 129P3/J) and one outbred (CD-1) strains across a wide range of previously determined effective MA doses (5, 35, 70, 100 mg/kg) and test times (1-4 h). MA produced significant dose-dependent and strain-dependent increases in food intake with strong responses noted in DBA/2J, outbred CD-1 and AKR/J mice. More limited dose-specific increases in food intake following MA occurred in C3H/HeJ, BALB/cJ, CBA/J, SJL/J, SWR/J and C57BL/6J mice. In contrast, MA failed to significantly increase food intake in A/J, C57BL/10J and 129P/3J mice. MA-induced food intake correlated significantly across strains only following the two highest doses, and intake following only the highest MA dose correlated significantly across strains with intake following only a moderate glucoprivic dose of 2-deoxy-d-glucose. Thus, these inter-strain differences suggest that lipoprivic (e.g., MA intake) and glucoprivic (e.g., 2-deoxy-d-glucose intake) responsivity operate via only partially overlapping genetic mechanisms of action. The demonstration of genotype-dependent variability in this lipoprivic response may provide the basis for the subsequent identification of trait-relevant genes. Show less

In spite of recent evidence showing the importance of DBY (DEAD-box RNA helicase Y) in spermatogenesis in human, the biologic role of its homolog Dby (also known as Ddx3y) in the mouse is less clear. The present study aims at characterizing the molecular structure of Dby and comparing its expression with its X- and autosome-linked homologs in embryonic gonads and developing germ cells in mice. Molecular cloning by rapid amplification of 3'-cDNA ends showed that the Dby gene in the mouse gives rise to 2 transcripts that differ only in the length of the 3'-untranslated region as a consequence of the use of alternative polyadenylation signals. Measurement by quantitative real-time polymerase chain reaction showed that both transcripts were ubiquitously expressed and were present in male germ cells and Sertoli cells. They were more abundant in type A spermatogonia compared with pachytene spermatocytes and round spermatids. Expression of Dby in the embryonic gonad increased from day 10.5 and reached a peak at day 17.5. The expression level of Dby decreased after birth and remained low in adult male gonads. Although the level of expression of Dby was much lower than its X chromosome homolog, Ddx3 (also known as Ddx3x) in all samples examined, the pattern of expression of the 2 genes was comparable. In contrast, their autosomal homolog, D1Pas1(also known as PL10), was predominantly expressed in pachytene spermatocytes and round spermatids. This result is in accord with meiotic sex chromosome inactivation in that Dby and Ddx are replaced in pachytene spermatocytes by their autosomal retroposon. These observations indicate that unlike DBY in humans, the role of Dby in spermatogenesis is less obvious in the mouse and its biologic activity may be replaced by that of Ddx3 and D1Pas1. Show less

NAD(P)H quinone oxidoreductase 1 (NQO1) is a ubiquitous flavoenzyme that catalyzes two-electron reduction of quinones to hydroquinones utilizing NAD(P)H as an electron donor. NQO1 binds and stabilizes several short-lived proteins including the tumor suppressors p53 and p73 and the enzyme ornithine decarboxylase (ODC). Dicoumarol is a widely used potent competitive inhibitor of NQO1 enzymatic activity, which competes with NAD(P)H for binding to NQO1. Dicoumarol also disrupts the binding of NQO1 to p53, p73, and ODC and induces their ubiquitin-independent proteasomal degradation. We report here the crystal structure of human NQO1 in complex with dicoumarol at 2.75 A resolution. We have identified the interactions of dicoumarol with the different residues of NQO1 and the conformational changes imposed upon dicoumarol binding. The most prominent conformational changes that occur in the presence of dicoumarol involve Tyr 128 and Phe 232 that are present on the surface of the NQO1 catalytic pocket. On the basis of the comparison of the NQO1 structure in complex with different NQO1 inhibitors and our previous analysis of NQO1 mutants, we propose that the specific conformation of Tyr 128 and Phe 232 is important for NQO1 interaction with p53 and other client proteins. Show less

Adult patients with idiopathic hypogonadotropic hypogonadism (IHH) typically present with absent puberty and therefore have prepubertal testes. IHH is recognized as one of the few curable causes of male infertility and is often effectively treated with either gonadotropins or pulsatile GnRH therapy. The objective of this study was to determine the structure of the testis prior to initiation of treatment. Eight adult IHH patients with prepubertal testes (<4 ml), with no previous gonadotropin therapy and with no history of cryptorchidism underwent open bilateral testicular biopsy prior to the initiation of hormonal treatment. The testes of all patients showed seminiferous cords separated by interstitium composed of blood vessels, connective tissue cells and collagen fibres but typical adult Leydig cells were absent. The cords contained only Sertoli cells and early type A spermatogonia. The spermatogonia mostly resided in the centre of the cords and were often large, typical of gonocytes. Sertoli cells appeared immature with ovoid nuclei devoid of infoldings and cytoplasm that lacked polarity. Tight junctional complexes commonly found connecting adult Sertoli cells were lacking. These results demonstrate that the immature testes from patients with the severe form of IHH possess early spermatogonia that could possibly reinitiate spermatogenesis with appropriate hormone stimulation. Therefore, the immature testis of this IHH subset resembles those of prepubertal boys and may provide important biologic and genetic insights into testicular development. Show less

Gene expression profiling was performed using the National Institute on Aging 15,000-cDNA microarray to reveal the differential expression pattern of 160 genes between meiotic pachytene spermatocytes and postmeiotic round spermatids of the mouse. Our results indicate that more genes are expressed in spermatids than in spermatocytes. Genes participating in cell cycle regulation and chromatin structure and dynamics are preferentially expressed in spermatocytes, while genes for protein turnover, signal transduction, energy metabolism, and intracellular transport are prevalent in spermatids. This suggests that a switch of functional requirement occurs when meiotic germ cells differentiate into haploid spermatids. Concordant expression patterns were obtained when quantitative real-time polymerase chain reaction was performed to verify the microarray data. Interestingly, the majority of the differentially expressed genes were underrepresented in mitotic type A spermatogonia, and they were preferentially expressed in the testis. Our results suggest that an even higher proportion of the mouse genome is devoted to male gamete development from meiosis than was previously estimated. We also provide evidence that underscores the advantage of using purified germ cells over whole testes in profiling spermatogenic gene expression to identify transcripts that demonstrate stage-specific expression patterns. Show less

Experimental handling stress (EHS) was applied to clinically asymptomatic farmed goldfish (Carassius auratus L.). EHS affected the gills and skin integrity of the fish and was accompanied by increased levels of plasma glucose, cortisol and interleukin-10 (IL-10). EHS application was followed by highly significant enhancement of the rate of infection with a virulent Aeromonas salmonicida isolate. Cumulative ulceration at the initial phase of the ensuing goldfish ulcerative disease (GUD) evidenced a facilitating role of EHS in the onset of GUD. Host susceptibility to the pathogen increased from 40% in unstressed fish to 90% in the stressed fish. A. salmonicida could be reisolated from the early-stage skin lesions only, whereas opportunistic strains, other than A. salmonicida (A. sobria and A. hydrophila), were recovered from progressive-stage ulcers. The implication of these findings in fish aquaculture is discussed. Show less

The overall goal of this study is to unravel the role(s) played by glial cell line-derived neurotrophic factor (GDNF) in the fate of spermatogonial stem cells. There is great interest in the biology of spermatogonial stem cells, or A(single) spermatogonia, because of their importance in the treatment of infertility, the development of contraceptives, and the understanding of the etiology of testicular cancer, particularly seminoma. In the mouse, spermatogonial stem cells express GFRalpha-1, the receptor for GDNF, and respond to this growth factor in vivo and in vitro. GDNF is produced by the adjacent Sertoli cells, which are part of the germ-line stem cell niche in vertebrates. We specifically isolated GFRalpha-1-positive spermatogonia using an immunomagnetic bead technique. We then stimulated the cells with 100 ng/mL of rGDNF for 10 hours; unstimulated cells served as negative controls. Microarray analysis, immunocytochemistry, and Western blotting revealed that Numb, a regulator of the Notch pathway, is upregulated by GDNF in spermatogonial stem cells. There are indications that in rats, mice, and humans, the Notch pathway promotes spermatogonial differentiation. We observed that an increase in Numb expression is concomitant with Notch degradation in these cells. Thus, through Numb, GDNF might inhibit differentiation and allows the maintenance of the stem cell pool in the mouse seminiferous epithelium. Show less

The principal goal of the Israel Structural Proteomics Center (ISPC) is to determine the structures of proteins related to human health in their functional context. Emphasis is on the solution of structures of proteins complexed with their natural partner proteins and/or with DNA. To date, the ISPC has solved the structures of 14 proteins, including two protein complexes. It has adopted automated high-throughput (HTP) cloning and expression techniques and is now expressing in Escherichia coli, Pichia pastoris and baculovirus, and in a cell-free E. coli system. Protein expression in E. coli is the primary system of choice in which different parameters are tested in parallel. Much effort is being devoted to development of automated refolding of proteins expressed as inclusion bodies in E. coli. The current procedure utilizes tagged proteins from which the tag can subsequently be removed by TEV protease, thus permitting streamlined purification of a large number of samples. Robotic protein crystallization screens and optimization utilize both the batch method under oil and vapour diffusion. In order to record and organize the data accumulated by the ISPC, a laboratory information-management system (LIMS) has been developed which facilitates data monitoring and analysis. This permits optimization of conditions at all stages of protein production and structure determination. A set of bioinformatics tools, which are implemented in our LIMS, is utilized to analyze each target. Show less

The objective of this study was to compare the expression of Col1a1, Col1a2, and procollagen I in the seminiferous tubules of immature and adult mice and to characterize the cellular expression pattern of procollagen I in germ cells during spermatogenesis in order to provide necessary groundwork for further functional studies in the process of spermatogenesis. Microarray analysis demonstrated that Col1a1 and Col1a2 were abundantly expressed in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice, and the expression levels of Col1a1 and Col1a2 mRNA were validated using a semi-quantitative RT-PCR assay. Western blot analysis further confirmed that procollagen I was expressed at a higher level in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice. Immunohistochemical analysis revealed that type A spermatogonia were positive for procollagen I in the testis of 6-day-old mice, whereas Sertoli cells were negative for this protein. The in vivo procollagen I staining in type A spermatogonia was corroborated in spermatogonia exhibiting a high potential for proliferation and the ability to form germ cell colonies in in vitro culture. Moreover, procollagen I was also detected in type A spermatogonia, intermediate spermatogonia, type B spermatogonia, and preleptotene spermatocytes in the adult mouse testes, but positive staining disappeared in more differentiated germ cell lineages detaching from the basement membrane, including leptotene spermatocytes, pachytene spermatocytes, round spermatids and elongated spermatids. These data suggest that Col1a1, Col1a2 and procollagen I are associated with type A spermatogonia and play a potential role in mediating the detachment and migration of germ cells during spermatogenesis. Show less

Mouse strain differences for intake of sucrose and saccharin have been reported across studies, and some of these differences have been related to variants of the Tas1r3 taste receptor gene. However, several methodological concerns remain, including use of relatively few strains and/or a limited number of palatable concentrations in previous analyses. The present study examined strain differences in sucrose intake among 11 inbred (A/J, AKR/J, BALB/cJ, CBA/J, C3H/HeJ, C57BL6/J, C57BL10/J, DBA/2J, SJL/J, SWR/J, 129P3/J) and one outbred (CD-1) mouse strains across nine different sucrose concentrations (0.0001-20%) using two-bottle 24-h preference tests which controlled for sucrose concentration presentation effects, sucrose and water bottle positions, and measurement of kilocalorie intake as sucrose or chow. A/J, C57BL/6J, CD-1 and SWR/J strains consumed the greatest (11.6-22 ml) amount of sucrose, whereas the A/J, C57BL/10J, SJL/J and SWR/J strains consumed the greatest (44-56%) percentages of kilocalories as sucrose. The AKR/J, CBA/J, C3H/HeJ and DBA/2J strains consumed the least (6.9-7.9 ml) amount of sucrose, and displayed lower (20-30%) percentages of kilocalories consumed as sucrose. Whereas A/J, C57BL/6J, C57BL/10J, CD-1, SWR/J and SJL/J strains all displayed the most pronounced compensatory decreases in chow intake as the percentage of kilocalories consumed as sucrose increased, the AKR/J, C3H/HeJ and DBA/2J strains failed to significantly alter chow intake even at high sucrose concentrations. There was a paucity of significant correlations in the percentage of sucrose intake between sucrose concentrations, but percentage of sucrose intake at lower concentrations did correlate with previous descriptions of saccharin intake and variants of the Tas1r3 taste receptor gene. These data demonstrate clear mouse strain differences across a range of measures in sucrose intake across a wide range of concentrations, but caution against extrapolating between extremely high and low concentrations. The identification of strains with diverging abilities to regulate kilocalorie intake when presented with high sucrose concentrations may lead to the successful QTL mapping of this trait. Show less

An unusual case is discussed in which a child presented with a significant facial deformity that was thought to be a soft tissue tumor. After surgical exploration it was found to be a fibrous encapsulated mass containing a foreign body. Show less

The identification and physical isolation of testis stem cells, a subset of type A spermatogonia, is critical to our understanding of their growth regulation during the first steps of spermatogenesis. These stem cells remain poorly characterized because of the paucity of specific molecular markers that permit us to distinguish them from other germ cells. Thus, the molecular mechanisms driving the first steps of spermatogenesis are still unknown. We show in the present study that GFR alpha-1, the receptor for GDNF (glial cell line-derived neurotrophic factor), is strongly expressed by a subset of type A spermatogonia in the basal part of the seminiferous epithelium. Using this characteristic, we devised a method to specifically isolate these GFR alpha-1-positive cells from immature mouse testes. The isolated cells express Ret, a tyrosine kinase transmembrane receptor that mediates the intracellular response to GDNF via GFR alpha-1. After stimulation with rGDNF, the isolated cells proliferated in culture and underwent the first steps of germ cell differentiation. Microarray analysis revealed that GDNF induces the differential expression of a total of 1124 genes. Among the genes upregulated by GDNF were many genes involved in early mammalian development, differentiation, and the cell cycle. This report describes the first isolation of a pure population of GFR alpha-1-positive cells in the testis and identifies signaling pathways that may play a crucial role in maintaining germ-line stem cell proliferation and/or renewal. Show less

In the mammalian testis, the germ line stem cells are a small subpopulation of type A spermatogonia that proliferate and ultimately differentiate into sperm under the control of both endocrine and paracrine factors. To study the early phases of spermatogenesis at the molecular level, an in vitro system must be devised whereby germ line stem cells can be either cultured for a prolonged period of time or expanded as cell lines. In the study reported here, we chose to immortalize type A spermatogonia using the Simian virus large T-antigen gene (LTAg) under the control of an ecdysone-inducible promoter. While the cells escaped the hormonal control after a finite number of generations and expressed the LTAg constitutively, their growth remained slow and the cells exhibited morphological features typical of spermatogonia at the light microscopic level. Moreover, the cells expressed detectable levels of protein markers specific for germ cells such as Dazl, and specific for germ line stem cells such as Oct-4, a transcription factor, and GFRalpha-1, the receptor for glial cell line-derived neurotrophic factor (GDNF). Further analysis confirmed the spermatogonial phenotype and also revealed the expression of markers expressed in stem cells such as Piwi12 and Prame11. Since the cells respond to GDNF by a marked increase in their rate of proliferation, this cell line represents a good in vitro model for studying aspects of mouse germ line stem cell biology. Show less

Protein-protein interactions are essential for life. Yet, our understanding of the general principles governing binding is not complete. In the present study, we show that the interface between proteins is built in a modular fashion; each module is comprised of a number of closely interacting residues, with few interactions between the modules. The boundaries between modules are defined by clustering the contact map of the interface. We show that mutations in one module do not affect residues located in a neighboring module. As a result, the structural and energetic consequences of the deletion of entire modules are surprisingly small. To the contrary, within their module, mutations cause complex energetic and structural consequences. Experimentally, this phenomenon is shown on the interaction between TEM1-beta-lactamase and beta-lactamase inhibitor protein (BLIP) by using multiple-mutant analysis and x-ray crystallography. Replacing an entire module of five interface residues with Ala created a large cavity in the interface, with no effect on the detailed structure of the remaining interface. The modular architecture of binding sites, which resembles human engineering design, greatly simplifies the design of new protein interactions and provides a feasible view of how these interactions evolved. Show less

The transcriptomes of mouse type A spermatogonia (Spga), pachytene spermatocytes (Spcy), and round spermatids (Sptd) were determined by sequencing the respective SAGE (Serial Analysis of Gene Expression) libraries. A total of 444,015 tags derived from one Spga, two Spcy, and one Sptd library were analyzed, and 34,619 different species of transcripts were identified, 5279 of which were novel. Results indicated the germ-cell transcriptome comprises of more than 30,000 transcripts. Virtual subtraction showed that cell-specific transcripts constitute 12-19.5% of the transcriptome. Components of the protein biosynthetic machinery are highly expressed in Spga. In Spcy transcription factors are abundantly expressed while transcripts encoding proteins involved in chromosome remodeling and testis-specific transcripts are prominent in Sptd. The databases generated by this work provide very useful resources for cellular localization of genes in silico. They are also extremely useful as sources for identification of splice variants of genes in germ cells. Show less

Inflammatory myofibroblastic tumor is a rare clinical entity that can first present in the oral cavity. Though considered a benign lesion, it can behave very locally and aggressively, with significant bony destruction. A rare case of inflammatory myofibroblastic tumor is presented, along with the surgical treatment and an overview of this interesting clinical lesion. Show less

A functionally important, interface domain between transmembrane segments (TMSs) IV and XI of the NhaA Na+/H+ antiporter of Escherichia coli has been unraveled. Scanning by single Cys replacements identified new mutations (F136C, G125C, and A137C) that cluster in one face of TMS IV and increase dramatically the Km of the antiporter. Whereas G125C, in addition, causes a drastic alkaline shift to the pH dependence of the antiporter, G338C alleviates the pH control of NhaA. Scanning by double Cys replacements (21 pairs of one replacement per TMS) identified genetically eight pairs of residues that showed very strong negative complementation. Cross-linking of the double mutants identified six double mutants (T132C/G338C, D133C/G338C, F136C/S342C, T132C/S342C, A137C/S342C, and A137C/G338C) of which pronounced intramolecular cross-linking defined an interface domain between the two TMSs. Remarkably, cross-linking by a short and rigid reagent (N,N'-o-phenylenedimaleimide) revived the Li+/H+ antiport activity, whereas a shorter reagent (1,2-ethanediyl bismethanethiosulfonate) revived both Na+/H+ and Li+/H+ antiporter activities and even the pH response of the dead mutant T132C/G338C. Hence, cross-linking at this position restores an active conformation of NhaA. Show less

Complementary DNA microarray and quantitative polymerase chain reaction were used as tools for discovering genes that are differentially expressed in the mouse under normal physiological conditions at distinctive stages of male germ cell development, that is, type A spermatogonia, pachytene spermatocytes, and round spermatids. By using this strategy, we identified a set of genes exhibiting differential expression patterns in spermatogenesis, suggesting that specific functions of the encoded products occurred during the developmental process. Among them were several genes previously not known to be active in testis, which signified undiscovered functional roles of these genes during spermatogenesis. Many of the genes identified were not previously characterized. This study highlights new targets for manipulation to unravel the molecular mechanism of spermatogenesis. Show less

Spermatogenesis is the process of differentiation of diploid type A spermatogonia to haploid spermatozoa. Several subtypes of A spermatogonia have been characterized in the adult mouse testis. These include A-single (A(s)), A-paired (A(pr)), A-aligned (A(al)), and A1-A4. However, in the immature testis, very little information is available on subtypes and morphological features of type A spermatogonia. Six-day-old mouse testes, fixed either in Bouin solution or 5% glutaraldehyde, were embedded in paraffin and Epon, respectively. Thick sections (approximately 1 microm) of Epon-embedded tissue were stained with toluidine blue and revealed three subtypes of spermatogonia by light microscopy. The smallest spermatogonia (subtype I) appeared as single cells and exhibited a round or oval flattened nucleus with one or two prominent dense nucleoli and a characteristic unstained round and centrally located vacuole. These cells bound toluidine blue more avidly and appeared darker in comparison with the other cell types. Electron microscopy of thin sections (90 nm) revealed a finely granulated chromatin homogeneously distributed in the nucleus and sparse organelles in the cytoplasm. The second subtype of spermatogonia (subtype II) also displayed dark staining but was larger than subtype I; there was no central vacuole in the nucleus and heterochromatin clumps were observed. The largest subtype of spermatogonia (subtype III) showed large heterochromatin clumps and a pale staining nucleus. Intercellular bridges were noted between subtypes II and III. Based on the dye avidity, the three subtypes were classified as dark, transitional, and pale spermatogonia, respectively. Image analyses of 30 different cells of each subtype revealed a decline in gray-scale intensity from subtype I to III. Five-micrometer sections of paraffin-embedded tissue were immunoassayed with an antibody against the glial cell-derived neurotrophic factor family receptor alpha-1 (GFRalpha-1) receptor, a putative marker for undifferentiated spermatogonia, showing positive reaction only in germ cells. The pattern of GFRalpha-1 expression, coupled to the overall morphology of the cells, indicates that at this stage of development, mouse seminiferous tubules contain essentially A(s), A(pr), and possibly A(al) spermatogonia. Thus, the present study indicates the presence of subtypes of type A spermatogonia in the immature mouse testis similar to that described previously in adult monkey and man. Show less

Overexpression of vascular endothelial growth factor (VEGF) in the testis of transgenic mice induces infertility, suggesting a potential role for VEGF in the process of spermatogenesis. Spermatogenesis occurs within the confines of the seminiferous tubules, and the seminiferous epithelium lining these tubules consists of Sertoli cells and germ cells in various stages of maturation. We investigated the source of VEGF and VEGF-target cells within the seminiferous tubules of the normal mouse testis. Sections of testes fixed in Bouin solution and embedded in paraffin were subjected to immunofluorescent staining with specific antibodies against VEGF, and its receptors, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1). Total RNA was extracted from isolated populations of Sertoli cells, type A spermatogonia, pachytene spermatocytes, and spermatids. Primer pairs specific for VEGF and its receptors were designed and reverse-transcriptase polymerase chain reaction (RT-PCR) was performed. Immunofluorescent studies indicated that VEGF is strongly expressed in the cytoplasm of Sertoli cells. VEGFR-1 and VEGFR-2 were not expressed by the Sertoli cell. In contrast, a differential expression of VEGF receptors was observed in germ cells. Although VEGFR-2 was expressed in the cytoplasm of type A spermatogonia, VEGFR-1 was expressed in the acrosomal region of spermatids and spermatozoa. Pachytene spermatocytes did not exhibit any staining. Further, we examined the transcription of VEGF and its receptors by RT-PCR. VEGF was actively transcribed only in Sertoli cells. The transcription of VEGFR-2 was confined to type A spermatogonia. Interestingly, VEGFR-1 was transcribed both in pachytene spermatocytes and round spermatids. The mRNA expression of VEGFR-1 and VEGFR-2 in germ cells was inversely correlated during postnatal development of the mouse testis. Thus, VEGF may play a potential role in regulating the initial stages of the process of spermatogonial proliferation through VEGFR-2 and spermiogenesis through VEGFR-1. Show less

Buccal cellulitis resulting from Haemophilus Influenzae type B (Hlb) is an uncommon yet potentially life-threatening illness that afflicts the facial soft tissues of the very young. Early recognition is essential for the effective treatment of this illness. A clinical case of Haemophilus Influenzae buccal cellulitis is presented, accompanied by a discussion of the presenting symptoms, diagnosis and treatment of this unusual childhood infection. Show less

Using differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) we identified transcripts encoding for the RNA helicase mDEAH9, Ran binding protein 5 (RanBP5), and 3 novel complementary DNAs designated GC3, GC12, and GC14 in developing testicular germ cells. Sources of RNA for the initial DDRT-PCR screen were purified mouse type A spermatogonia, adult mouse wild-type testis, and W/W(v) mutant mouse testis. We identified cDNA fragments for mDEAH9, RanBP5, GC3, GC12, and GC14 in testis and type A spermatogonia samples from wild-type mice, but not in samples from the W/W(v) mouse testis. These same transcripts were absent in Northern blots of testis RNA from mice treated with busulfan 30 days prior, but were present in testis RNA from wild-type mice at 5, 15, 25, and 40 days of age. The mDEAH9 gene was expressed in many tissues, whereas RanBP5 and GC12 genes were expressed predominantly in the testis with much lower expression in other tissues. The expression of GC3 and GC14 were limited to the testis as evidenced by Northern blot and RT-PCR analyses. The mDEAH9 transcript was not detected in cultured interstitial cells but was found at low levels in cultured immature Sertoli cells, whereas the RanBP5, GC3, GC12, and GC14 transcripts were not detected in either cultured testicular interstitial cells or cultured Sertoli cells. RT-PCR analyses of isolated spermatogonia, pachytene spermatocytes, and round spermatids revealed that mDEAH9, RanBP5, GC3, GC12, and GC14 genes were expressed in all 3 cellular populations. In situ hybridization analyses of testis samples from 40-day-old mice localized expression of mDEAH9, RanBP5, GC3, GC12, and GC14 to the seminiferous tubules. RanBP5 expression appeared to be regulated during the cycle of the seminiferous epithelium, with the highest expression in stages III through VII. Expression of GC14 was greatest in the meiotic germ cell populations. Show less

SP1 (108 amino acids) is a boiling-stable stress-responsive protein. It has no significant sequence homology to other stress-related proteins or to small heat-shock proteins (sHsps). SP1 activity is ATP-independent, similar to other small heat-shock proteins. Based on these features, it is expected that the structure-function relationship of SP1 will be unique. In this work, the crystallization and preliminary crystallographic data of native SP1 and its selenomethionine derivative are described. Recombinant SP1 and its selenomethionine derivative were expressed in Escherichia coli and used for crystallization experiments. SP1 crystals were grown from 0.1 M HEPES pH 7.5, 20% PEG 3K, 0.2 M NaCl. One to four single crystals appeared in each droplet within a few Days and grew to dimensions of about 0.5 x 0.5 x 0.8 mm after about two weeks. Diffraction studies of these crystals at low temperature indicated that they belong to space group I422, with unit-cell parameters a = 89, b = 89, c = 187 A. Efforts to crystallize the selenomethionine derivative of SP1 are in progress. Show less

Sertoli cells isolated from 6-day postpartum mouse testes were conditionally immortalized with the simian virus 40 large tumor antigen gene (SV40-LTAg) under the control of a promoter inducible with ponasterone A, an analog of ecdysone. This strategy produced 2 cell lines, which exhibited mixed phenotypes. We first tested the conditional expression of the LTAg gene in the presence or absence of ponasterone A. The results showed that both cell lines expressed LTAg when the inducer was present in the culture media. When ponasterone A was removed, the majority of the cells died. After 60 generations, however, the continued expression of LTAg in the absence of the hormone indicated that unknown changes may have occurred in the genome of the cells. One of the cell lines was further subcloned, resulting in 7 new lines exhibiting a morphology resembling that of Sertoli cells in tissue culture. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on RNA collected from each cell line in order to determine which cells were phenotypically similar to Sertoli cells in vivo. All cell lines expressed the products of the Sertoli cell-specific genes stem cell factor (SCF) and sulfated glycoprotein-2 (SGP-2), in addition to alpha-inhibin, GATA-1, and steroidogenic factor-1. Further, the lines express growth and differentiation factors known to act upon germ cells in vivo and in vitro such as leukemia inhibitory factor (LIF), transforming growth factor beta (TGF-beta), and basic fibroblast growth factor (bFGF). Moreover, when used as feeder layers in cocultures, at least 2 of these lines are able to maintain the viability of type A spermatogonia for at least 7 days and to support the first steps of spermatogonial differentiation. Show less