Properties and compartmentalization of the testicular receptor for 1,25-dihydroxyvitamin D3

Adult rat testis contains a specific, high-affinity, low-capacity binding protein for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) with properties similar to 1,25-(OH)2D3 receptors in other tissues. The receptor sediments at 3.5 +/- 0.2 S20,w in high-salt sucrose density gradients, but aggregates in low-salt gradients. Binding of 1,25-(OH)2D3 was abolished by trypsin, but not by DNase or RNase. Binding was also heavily reduced by the sulfhydryl alkylating agent, N-ethylmaleimide, and by the mercurial reagent, mersalyl, showing that free, reduced SH-groups are necessary for hormone-binding activity. The receptor shows high affinity for 1,25-(OH)2D3 (Kd = 3 X 10(-11) M), but low capacity (Nmax = 8 fmol/mg protein) and is specific for 1,25-(OH)2D3 (Affinity: 1,25-(OH)2D3 greater than 1,24(R),25-(OH)3D3 greater than 25-OH-D3 greater than 1 alpha-OH-D3 greater than 24(R),25-(OH)2D3 much greater than 17 beta-estradiol, testosterone, dexamethasone, R5020, progesterone). With 0.6 nM [3H]1,25-(OH)2D3 and at 0 degrees C, maximum specific binding was achieved after 4 h, and the occupied receptors were stable for more than 24 h. The dissociation of hormone-receptor complexes was temperature-dependent and very slow at low temperature (t1/2 (0 degrees C) much greater than 48 h). At 0 degrees C, the second order association rate constant and the pseudo-first order dissociation rate constant were 2.7 X 10(7) M-1 min-1 and 2 X 10(-5) min-1, respectively. Receptors for 1,25-(OH)2D3 are present in similar amounts in isolated seminiferous tubules and interstitial tissue of adult rats. No specific binding of [3H]1,25-(OH)2D3 could be detected in cultured immature Sertoli cells, cultured immature peritubular (myoid) cells or crude germ cells.     

Protoplast transformation of group N streptococci with cryptic plasmids

Abstract By using an extension to group N streptococci of a contransformation procedure we have introduced 4 different-sized cryptic plasmids for Streptococcus lactis into the plasmid-free S. lactis IL1403. A mixture of 4 cryptic plasmids with an indicator plasmid (pHV1301) conferring erythromycin resistance was used for IL1403 protoplast transformation. Under such conditions, 41.5% of the erythromycin-resistant transformants were contransformed with one of the cryptic plasmids in addition to pHV1301. Indicator plasmid pHV1301 was later spontaneously segregated from doubly transformed cells. This protocol should be very useful for constructing lactic streptococcal strains bearing any phenotypically cryptic plasmid.     

Phytoplankton population dynamics of a small reservoir: physical/biological coupling and the time scales of community change

This paper describes the day-to-day changes in species compositionthat resulted from intermittent wind mixing of the surface watersof a small reservoir. Two major scales of community change weredetected: a short-term (>1 day) scale associated with theredistribution of cells within the basin, and a 5–14-dayscale associated with growth responses. The physical scalesof change were found to be almost identical to the biologicalscales: wind stress caused changes in the temperature gradientof surface waters at scales of a day, and major vertical mixingevents occurred at scales of 10–14 days. The presenceof buoyant species ensured that rapid advection of populationsfollowed wind events. Community change was a function of bothadvection and growth, so that both real and apparent changesin abundance occurred. The observed seasonal succession wasboth a true succession and a changing sequence of populationsdependent on horizontal advection of water within the basin.Consistent stratification was present throughout the summerperiod and phytoplankton diversity was low. Even so, the seasonalsuccession was best described as a series of allogenic perturbationsfollowed by biological restructuring of the community. Dailysampling was necessary to document fully the mechanisms drivingthe seasonal succession of species.     

Primary astroglial cultures

Conclusion Primary cultures from neonatal rat brain consist mainly of astroglial cells, immunohistochemically identified by GFAp and S-100. As other cells than astrocytes may survice in the culture, specific markers for the expected cells were used. Cells with phagocytic properties, endothelial-like cells, oligoblasts, ependymal cells and mesenchymal cells were identified. No neurons have so far been detected.The astroglial cells have a high-affinity uptake for glutamate, aspartate GABA, taurine and hypotaurine, while there is probably a non-saturable uptake of norepinephrine, dopamine and 5-HT. The enzymes MAO, COMT, GABA-T and GS have been demonstrated. It thus seems that astrocytes take part in the inactivation of neurotransmitters, although amino acids and monoamines are taken up with different mechanisms.The presence of receptors for different neurotransmitters and neuromodulators has been demonstrated on astrocytes.Astroglial-enriched cultures from various brain regions have shown that the cells express specialized functional properties concerning neurotransmitter uptake, metabolizing enzymes and receptor density.Astroglial cell differentiation in culture is shortly reviewed and one possibility to affect this maturation by co-cultivation with neuronal containing cultures is point out.     

Ultrastructure of the surface of the epithelial cells in the rat retina

Summary The epithelial cells in the retina of rats were examined by scanning and transmission electron microscopy.The apical surface of the epithelial cells is covered by a large number of microvilli, which are embedded in an extracellular material. Distinct holes remaining after detached photoreceptor cell segments are numerous. The cells are polyhedronal, usually hexagonal and holes are scattered in their plasma membrane. Many of the epithelial cells are binucleated. The borders between adjacent cells are always close to each other. Numerous cytoplasmic folds or foot processes occur at the base of the cells as well as an extracellular space.The characteristic surface structures are discussed in relation to the function of the retinal epithelial cells.Supported by grants from the Swedish Medical Research Council (B70-12X-2543-02) and Riksföreningen mot Cancer (69171, 265-B69-01X).     

Human arterial smooth muscle cells in culture: Inverse relationship between proliferation and expression of contractile proteins

Summary Human arterial smooth muscle cells (hASMC) from explants of the inner media of uterine arteries were studied in secondary culture. We had previously found that these cells depend on exogenous platelet-derived growth factor (PDGF) for proliferation in vitro. Deprivation of the serum mitogen(s) by culture in plasma-derived serum or bovine serum albumin (BSA) caused a true growth arrest that was reversible upon reexposure to the mitogen(s). When added to serum-containing medium, heparin caused a reversible growth arrest which could be competed for by increasing concentrations of serum. In the current study we used a set of smooth muscle-specific actin and myosin, antibodies to study the expression of contractile proteins in stress fibers under indirect immunofluorescence on hASMC in culture. Even in sparse culture, grwoth-arrested hASMC expressed stress fibers containing these actin and myosin epitopes. This was true irrespective of whether growth arrest was achieved by culture in media containing only BSA or a combination of heparin and whole blood serum. hASMC proliferating in whole blood serum in sparse culture did not express such strees fibers, as judged by immunofluorescent staining. This was true also for cells that were restimulated to proliferate in serum after a growth arrest. Utilizing a monoclonal antibody against a nuclear antigen expressed in proliferating human cells, we were able to demonstrate an inverse relationship between the expression of this antigen and the SMC-specific contractile proteins, respectively. Under these culture conditions, the reversible transition between defifferentiated and differentiated hASMC was almost complete and terminated about 1 wk after the change in culture condition. We conclude that hASMC in vitro respond, to exogenous PDGF by proliferation and dedifferetiation as a single population of cells. We also conclude that this modulation is reversible, because the cells become uniformly quiescent and differentiated when the mitogenic stimulus is blocked or removed. This study was supported by grants from the Swedish Medical Research Council (Project no. 4531 and 6816), the Swedish Association against Heart and Chest Diseases, the King Gustaf V and Queen Victoria Foundation, the National Institutes of Health, Bethesda, MD (grant HL 29873) and the Swedish National Board for Laboratory Animals.     

Untreated asymptomatic bacteriuria in girls: II--Effect of phenoxymethylpenicillin and erythromycin given for intercurrent infections.

OBJECTIVE--To investigate the effects of phenoxymethylpenicillin and erythromycin on urinary isolates from patients with untreated asymptomatic bacteriuria. DESIGN--Retrospective study of subgroup of patients from cohort followed up till the end of 1986. SETTING--Outpatient clinic for children with urinary tract infections. PATIENTS--51 Girls aged under 15 with untreated asymptomatic bacteriuria. INTERVENTIONS--Before 1982 intercurrent infections (mostly tonsillitis or otitis) were treated with phenoxymethylpenicillin; after 1982 erythromycin treatment was preferred. END POINTS--Change of bacterial strain in urinary tract and symptomatic recurrences of disease. MEASUREMENTS AND MAIN RESULTS--Bacteria identified by serotype and electrophoretic type and compared before and after antibiotic treatment. Bacteriuria eradicated and replaced by new strains in most patients treated with phenoxymethylpenicillin, leading to symptomatic recurrences in about 15%. Conversely, patients given erythromycin rarely showed change in bacteriuria and none suffered symptomatic recurrence. CONCLUSIONS--In girls with untreated asymptomatic bacteriuria the use of phenoxymethylpenicillin for intercurrent infections may lead to a change of urinary bacteria and leave them at substantial risk of acute pyelonephritis. With erythromycin this risk is small (2/20 courses in this series).     

Untreated asymptomatic bacteriuria in girls: I--Stability of urinary isolates.

OBJECTIVE--To assess the frequency of spontaneous changes of bacterial strains in patients with untreated asymptomatic bacteriuria. DESIGN--Retrospective analysis of samples from all patients with renal scarring and random sample of patients with normal kidneys. SETTING--Outpatient clinic for children with urinary tract infections. PATIENTS--54 Girls aged 3.3-15.5 years with untreated asymptomatic bacteriuria caused by Escherichia coli. INTERVENTION--None. END POINT--Change in bacterial strain. MEASUREMENTS AND RESULTS--Serotyping and electrophoretic analysis of sequential bacterial isolates, representing 151 patient years of untreated asymptomatic bacteriuria. A total of 24 changes of strain were identified. Eleven were related to medical interference such as treatment of other infections with antibiotics. CONCLUSIONS--Spontaneous changes of strain were uncommon, one change in 11.6 patient years, and thus are not a characteristic feature of the course of asymptomatic bacteriuria.     

A novel sialidase which releases 2,7-anhydro-alpha-N-acetylneuraminic acid from sialoglycoconjugates

The leech (Macrobdella decora) was found to contain two sialic acid-cleaving enzymes: an ordinary sialidase and a novel sialic acid-cleaving enzyme. This novel enzyme released 2,7-anhydro-alpha-N-acetylneuraminic acid (Neu2,7-anhydro5Ac) instead of alpha-N-acetylneuraminic acid (Neu5Ac) from 4-methylumbelliferyl-Neu5Ac, glycoproteins, and gangliosides. We have partially purified this novel sialidase from M. decora. We have also isolated Neu2,7-anhydro5Ac released from 4-methylumelliferyl-Neu5Ac and whale nasal keratan sulfate in pure form. The novel sialidase produced Neu2,7-anhydro5Ac only from sialoglycoconjugates, but not from free Neu5Ac. The structure of Neu2,7-anhydro5Ac produced by the novel sialidase was established by chemical analysis, mass spectrometry, and NMR spectroscopy. NMR analysis showed that instead of the original 2C5 conformation, the pyranose ring of Neu2,7-anhydro5Ac was in the 5C2 conformation, which makes the formation of the 2,7-anhydro bridge possible.     

Production of TNF-alpha and TNF-beta by staphylococcal enterotoxin A activated human T cells

Staphylococcal enterotoxin at concentrations of less than 1 pg/ml induces significant TNF activity in human peripheral blood T cells and monocytes. Maximal TNF activity is routinely detected after 48 to 72 h of culture. IL-2 and IL-4 were both growth promoting for human T cells but only IL-2 could efficiently induce TNF production. The production of TNF-alpha and TNF-beta differed greatly in kinetics. An early intracytoplasmatic production of TNF-alpha after 6 h was detected in both monocytes and T cells whereas a late production of TNF-beta (lymphotoxin) after 48 h, occurred in the T cell population. Induction of TNF-alpha and TNF-beta production by Staphylococcal enterotoxin requires the presence of both monocytes and T cells. The CD4+45R- but not CD4+45R+ and CD8+ cells supported TNF-alpha production in monocytes. The main lytic component from Staphylococcal enterotoxin-activated mononuclear cells is TNF-beta. CD4+ and CD8+ T cells produced about equal amounts of biologically active TNF into the culture supernatants but a fourfold higher frequency of TNF-beta producing cells was demonstrated among CD4+ vs CD8+ cells. The CD4+45R- T cell subset was an efficient producer of TNF-beta and IFN-gamma whereas the CD4+45R+ T cell subset produced significant amounts of TNF-beta but only marginal amounts of IFN-gamma.