Expression of the androgen receptor and 5α-reductase type 2 in the developing human fetal penis and urethra

Normal penile development is dependent on testosterone, its conversion via steroid 5 alpha-reductase type 2 to dihydrotestosterone, and a functional androgen receptor (AR). The goal of this study was to investigate the distribution of AR and 5 alpha-reductase type 2 in the developing human fetal external genitalia with special emphasis on urethra formation. Twenty fetal genital specimens from normal human males (12-20 weeks gestation) were sectioned serially and stained by avidin-biotinylated peroxidase complex method with antigen retrieval. Stained sections throughout male genital development documented the expression of AR and 5 alpha-reductase type 2 in the phallus. Between 12 and 14 weeks of gestation, AR was localized to epithelial cells of the urethral plate in the glans, the tubular urethra of the penile shaft, and stromal tissue surrounding the urethral epithelium. In the fetal penis between 16 and 20 weeks gestation, the density of AR expression was greatest in urethral epithelial cells versus the surrounding stromal tissues. There was a characteristic pattern of AR expression in the glandular urethral epithelium between 16 and 20 weeks gestation. AR expression was greater along the ventral aspect of the glandular urethra than along the dorsal aspect of the urethral epithelium. The expression of 5 alpha-reductase type 2 was localized to the stroma surrounding the urethra, especially along the urethral seam area in the ventral portion of the remodeling urethra. These anatomical studies support the hypothesis that androgens are essential for the formation of the ventral portion of the urethra and that abnormalities in either the AR or 5 alpha-reductase type 2 can explain the occurrence of hypospadias.     

Role of warm stratification in promoting germination of seeds of Empetrum hermaphroditum (Empetraceae), a circumboreal species with a stony endocarp

The broad objective of this research was to define the role of warm (≥15°C) stratification in breaking dormancy in seeds with stony endocarps that require warm-plus-cold (～0°-10°C) stratification for germination. This question was addressed using seeds (true seed + endocarp, hereafter called seeds) of Empetrum hermaphroditum. Only 2-5% of freshly matured seeds collected in September and October at five sites in Sweden germinated in light at daily alternating temperature regimes of 15°/6°, 20°/10°, and 25°/15°C. Dormancy was not due to impermeability of the stony endocarp surrounding each seed, and embryos did not grow prior to radicle emergence. Thus, seeds did not have physical, morphological, or morphophysiological dormancy. Long periods of either cold stratification (20 or 32 wk) or warm stratification (16 wk) resulted in a maximum of 22-38 and 10% germination, respectively, in light at 25°/15°C. After 12 wk warm stratification plus 20 wk cold stratification, 83-93% of the seeds germinated in light at the three temperature regimes. For a cold stratification period of 20 wk, germination increased with increase in length of the preceding warm stratification treatment. Gibberellic acid (GA(3)) promoted germination of 77-87% of the seeds. Based on dormancy-breaking requirements and response to GA(3), 62-78% of the seeds had intermediate physiological dormancy; the others had nondeep physiological dormancy. Contrary to suggestions of several other investigators that warm stratification is required to make the endocarp permeable to water via its breakdown by microorganisms, our results with E. hermaphroditum show that this is not the case. In this species, warm stratification is part of the dormancy-breaking requirement of embryos in seeds with intermediate physiological dormancy.     

Aluminum rapidly depolymerizes cortical microtubules and depolarizes the plasma membrane: evidence that these responses are mediated by a glutamate receptor

Efforts to understand how plants respond to aluminum have focused on describing the symptoms of toxicity and elucidating mechanisms of tolerance; however, little is known about the signal transduction steps that initiate the plant's response. Here, we image cortical microtubules and quantify plasma-membrane potential in living, root cells of intact Arabidopsis seedlings. We show that aluminum depolymerizes microtubules and depolarizes the membrane, and that these responses are prevented by calcium channel blockade. Calcium influx might involve glutamate receptors, which in animals are ligand-gated cation channels and are present in the Arabidopsis genome. We show that glutamate depolymerizes microtubules and depolarizes the plasma membrane. These responses, and also the inhibition of root elongation, occur within the first few min of treatment, but are evoked more rapidly by glutamate than by aluminum. Microtubule depolymerization and membrane depolarization, induced by either glutamate or aluminum, are blocked by a specific antagonist of ionotropic glutamate receptors, 2-amino-5-phosphonopentanoate; whereas an antagonist of an aluminum-gated anion channel blocks the two responses to aluminum but not to glutamate. For growth, microtubule integrity, and membrane potential, responses to combined glutamate and aluminum were not greater than to glutamate alone. We propose that signaling in response to aluminum is initiated by efflux of a glutamate-like ligand through an anion channel and the binding of this ligand to a glutamate receptor.     

The Arabidopsis thaliana PPX/PP4 phosphatases: molecular cloning and structural organization of the genes and immunolocalization of the proteins to plastids

The PPX/PP4 Ser/Thr protein phosphatases belong to the type 2A phosphatase subfamily and are present in most eukaryotic organisms. We have previously isolated two closely related DNAs encoding PPX isoforms (PPX-1 and PPX-2) of Arabidopsis thaliana. Here we report the molecular cloning of the genes encoding these proteins. The genes PPX-1 and PPX-2 are composed of eight exons and seven introns located at equivalent positions related to the coding sequences. Whereas the intron-exon organization of the PPX genes is completely different from that of the PP2A-3/PP2A-4 A. thaliana family, specific intron-exon boundaries are conserved among PPX genes from distantly related organisms. Based on GUS expression, both PPX genes show the same spatial and temporal pattern of expression: they are expressed in all the organs and tissues analyzed, and from the earliest stage of development. When PPX proteins were localized to the root in semi-thin methacrylate sections by immunofluorescence, staining was predominantly confined to small organelles, shown to be plastids by co-localization of PPX and ferredoxin. Interestingly, only some ferredoxin-positive plastids were also PPX-positive, and PPX staining was consistently brighter in the epidermis. The localization was confirmed with immunogold and electron microscopy. Our results suggest that, despite its strong sequence conservation, PPX in plants functions differently than in animals.     

GERMINATION ECOPHYSIOLOGY OF HYDROPHYLLUM APPENDICULATUM,A MESIC FOREST BIENNIAL

In north central Kentucky, seeds of the mesic forest biennial Hydrophyllum appendiculatum Michx., are innately dormant at maturity in June. Under natural and simulated seasonal temperature changes, dormancy break occurred in two stages. Root dormancy was broken by high summer temperatures, and shoot dormancy was broken by low winter temperatures. Consequently, roots emerged from seeds during autumn, and cotyledons emerged the following spring. A 90-day warm (30/15 C) stratification treatment broke root dormancy, but the roots emerged only after transfer to lower temperatures. After the warm stratification treatment, roots emerged from 93, 73, 6 and 9% of the seeds incubated at 5, 15/6, 20/10 and 30/15 C (12/12 hr), respectively. Zero, 28, 56 and 84 days of cold (5 C) stratification of seeds with emerged roots resulted in 9, 21, 49 and 82% cotyledon emergence, respectively, at 20/10 C. Thus, H. appendiculatum exhibits a type of morpho-physiological dormancy known as epicotyl dormancy. Although many seeds germinate the first year, others remain dormant and germinate in successive years until the fourth season after ripening.     

A new type of specialized morphophysiological dormancy and seed storage behaviour in Hydatellaceae, an early-divergent angiosperm family

Background and Aims

Recent phylogenetic analysis has placed the aquatic family Hydatellaceae as an early-divergent angiosperm. Understanding seed dormancy, germination and desiccation tolerance of Hydatellaceae will facilitate ex situ conservation and advance hypotheses regarding angiosperm evolution.

Methods

Seed germination experiments were completed on three species of south-west Australian Hydatellaceae, Trithuria austinensis, T. bibracteata and T. submersa, to test the effects of temperature, light, germination stimulant and storage. Seeds were sectioned to examine embryo growth during germination in T. austinensis and T. submersa.

Key Results

Some embryo growth and cell division in T. austinensis and T. submersa occurred prior to the emergence of an undifferentiated embryo from the seed coat (‘germination’). Embryo differentiation occurred later, following further growth and a 3- to 4-fold increase in the number of cells. The time taken to achieve 50 % of maximum germination for seeds on water agar was 50, 35 and 37 d for T. austinensis, T bibracteata and T. submersa, respectively.

Conclusions

Seeds of Hydatellaceae have a new kind of specialized morphophysiological dormancy in which neither root nor shoot differentiates until after the embryo emerges from the seed coat. Seed biology is discussed in relation to early angiosperm evolution, together with ex situ conservation of this phylogenetically significant group.     

Dynamic light scattering characterization of the detergent-free,delipidated (Ca2+ + Mg2+)-ATPase from sarcoplasmic reticulum

Dynamic light scattering studies have been conducted on the delipidated and detergent-removed (Ca²⁺ + Mg²⁺)-ATPase protein assemblies. Specific characterization of the state of aggregation and the extent of conformation change upon delipidation and detergent removal has been made. The results show that the prominent species are dimers and tetramers of very globular nature, with axial ratios of less than 2 : 1. The hydrodynamic radii of the dimers and the tetramers are, respectively, 57.5 Å and 74.5 Å.The globular nature of these observed entities differ from the delipidated ATPase proteins recently obtained (LeMaire, M., Jorgensen, K.E., Roigaard-Petersen, H. and Moller, J.V. (1976) Biochemistry 15, 5805–5812). Present results suggest that upon the removal of detergents from the lipid-free ATPase protein assembly, only a rather limited degree of aggregation takes place. Such a condition is consistent with models of the membrane protein system which has limited regions of hydrophobic contact. Oligomeric assemblies with aqueous channels is a possible active Ca²⁺ transport model consistent with results of the present data, as well as the data from several other recent studies.     

Kinesin force generation measured using a centrifuge microscope sperm-gliding motility assay.

To measure force generation and characterize the relationship between force and velocity in kinesin-driven motility we have developed a centrifuge microscope sperm-gliding motility assay. The average (extrapolated) value of maximum isometric force at low kinesin density was 0.90 +/- 0.14 pN. Furthermore, in the experiments at low kinesin density, sperm pulled off before stall at forces between 0.40 and 0.75 pN. To further characterize our kinesin-demembranated sperm assay we estimated maximum isometric force using a laser trap-based assay. At low kinesin density, 4.34 +/- 1.5 pN was the maximum force. Using values of axoneme stiffness available from other studies, we concluded that, in our centrifuge microscope-based assay, a sperm axoneme functions as a lever arm, magnifying the centrifugal force and leading to pull-off before stall. In addition, drag of the distal portion of the axoneme is increased by the centrifugal force (because the axoneme is rotated into closer proximity to the glass surface) and represents an additional force that the kinesin motor must overcome.     

Interactions between Mycobacterium leprae and simian immunodeficiency virus (SIV) in rhesus monkeys

Groups of rhesus monkeys were inoculated with: 1) simian immunodeficiency virus (SIV)B670 alone; 2) Mycobacterium leprae alone; 3) SIV plus M. leprae on the same day; and 4) M. leprae 2 weeks after SIV. Animals were monitored at intervals for virus loads, antibody responses to M. leprae glycolipid antigens and to SIV Gp120, T-cell CD4+ and CD4+ CD29+ subset percentages, leprosy and acquired immunodeficiency syndrome (AIDS) clinical symptoms. Five out of six animals developed leprosy in each co-inoculated group, compared to one out of six in the M. leprae-only-inoculated group, indicating that M. leprae/SIV co-infection increases the susceptibility to leprosy, regardless of the timing of the two infections. Animals in the co-infected group that received M. leprae 2 weeks after SIV had a significantly slower rate of AIDS progression and long-term survival was significantly greater (three out of six) compared to the group inoculated with SIV alone (zero out of seven). All M. leprae-only-inoculated animals (six out of six) survived. Post-SIV-inoculation, a rapid decrease in the percentages of CD4 + and CD4 + CD29 + T-cells was observed in the SIV-only-inoculated group that was significantly blocked by co-inoculation with M. leprae 2 weeks after SIV, but not by SIV on the same day. The virus load set point was increased by approximately two logs in the group inoculated with M. leprae and SIV on the same day compared to SIV 2 weeks prior to M. leprae or the SIV-only-inoculated group. The results indicate that M. leprae, inoculated 2 weeks after SIV, decreased the pathogenicity of SIV compared to inoculation of M. leprae and SIV on the same day or SIV alone. The decreased pathogenicity correlated with a diminished loss of CD4 + and CD4 + CD29 + T-cell subsets in the group inoculated with M. leprae 2 weeks after SIV compared to the group inoculated with SIV alone. IgG antibody responses to M. leprae-specific cell wall phenolic glycolipid-I antigen were inhibited by 2-week-prior or same-day SIV co-inoculation compared to M. leprae-only inoculated animals. The IgG anti-lipoarabinomannan antibody response was enhanced in the group inoculated with M. leprae and SIV on the same day compared to the groups inoculated with M. leprae alone or SIV 2 weeks prior to M. leprae. Antibody responses to SIV Gp120 antigen were unimpaired in both co-inoculated groups compared to SIV-only-inoculated groups. The antibody results show that the immune responses to SIV and M. leprae are interrelated in SIV/M. leprae co-infected animals.     

Further characterization of the binding of human recombinant interleukin 2 to heparin and identification of putative binding sites

We have previously provided compelling evidence that human recombinant interleukin 2 (IL-2) binds to the sulfated polysaccharides heparin, highly sulfated heparan sulfate and fucoidan. Here we show that IL-2 binding is dependent on heparin chain length, but with fragments as small as 15-mers retaining binding activity. The addition of exogenous heparin has no effect on the in vitro biological activity of IL-2. In addition soluble IL-2 receptor alpha and beta polypeptides do not compete with heparin for the binding of IL-2. IL-2 bound by heparin is still recognized by two IL-2 specific monoclonal antibodies, 3H9 and H2- 8, whose epitopes lie in the amino terminal region. Murine IL-2 unlike its human counterpart fails to bind to heparin. Human IL-2 analogs with single amino acid substitutions at positions Lys43, Thr51, and Gln126 analogs no longer bind to heparin. By contrast the Arg38Ala analog retains heparin full heparin binding activity. These experimental findings together with molecular modeling studies suggest two putative heparin binding sites on human IL-2, one involving four basic residues, Lys48, Lys49, Lys54, and His55, and the other being a discontinuous site comprising Lys43, Lys64, Arg81, and Arg83. Neither of these two clusters is completely conserved in murine IL-2. Overall our data suggest that the binding of human IL-2 to heparin and heparan sulfate does not interfere with IL-2/IL-2 receptor interactions. Therefore, binding to glycosaminoglycan may be a mechanism for retaining the cytokine in an active form close to its site of secretion in the tissue, thus favoring a paracrine role for IL-2.