Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy.

In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV-pulsed mature human dendritic cells.

[Pregabalin and gabapentin in multiple sclerosis: clinical experiences and therapeutic implications]. / Ionenkanalmodulatoren bei Multipler Sklerose : Bestandsaufnahme der Anwendung in der Praxis.

BACKGROUND: Due to a plethora of additional symptoms patients with multiple sclerosis (MS) receive symptomatic treatment besides disease-modifying therapies. Among the substances which are commonly used are ion channel modulators (e. g. pregabalin, gabapentin, carbamazepine). The aim of this study was to investigate the use of these drugs in clinical practice in a larger patient cohort. PATIENTS: Data from 533 MS patients [439 without and 94 patients with add-on therapy (treatment group)] were evaluated retrospectively. All patients received a detailed neurological examination including evaluation of EDSS scores. RESULTS: Pregabalin and gabapentin are used most commonly. Abnormal sensations are the most frequent reason for therapy initiation. Patients with higher EDSS values and/or under mitoxantrone treatment most frequently receive additional therapy. CONCLUSION: So far, it is not known whether the investigated agents exert a beneficial influence on the disease course of MS itself beyond a mere symptomatic treatment. Further research efforts and clinical studies are necessary to address this question.

Hexagonal magnetite nanoprisms: preparation, characterization and cellular uptake.

The capacity of iron oxide nanocrystals to heat tissue when subjected to an alternating magnetic field (AMF hyperthermia) is shape-selective. Although iron oxide nanostructures with numerous shapes have been synthesized to date, hexagonal Fe3O4 prisms of low toxicity remained elusive. Here, we report the use of a dual ligand system permitting feasible reaction conditions to synthesize nearly perfect hexagonal Fe3O4 nanoplatelet structures, with edge length of 45 ± 5 nm and thickness of 5 to 6 nm. Their Specific Absorption Rate (SAR) is >750 W g(Fe)-1. The Fe3O4 hexagons were coated with a dopamine-based ligand to increase dispersibility in aqueous buffers. The Fe3O4 hexagons were only minimally toxic to RAW264.7 cells, which can be utilized in cell-based cancer targeting approaches.

Ontogeny of catecholamine turnover rates in limbic and hypothalamic structures in relation to serum prolactin and gonadotropin levels.

Norepinephrine (NE) and dopamine (DA) concentrations and turnover rates have been studied in the n. accumbens, medial preoptic area (MPO) and anterior and posterior parts of the mediobasal hypothalamus of developing rats. Turnover rates are determined by injection of alpha-methyl-p-tyrosine 30 and 90 min prior to decapitation. NE concentrations and turnover in the n. accumbens were low in all age groups with slightly increased values between days 20 and 35 after birth whereas DA concentrations and turnover rates were low at day 15 and 20 and at high adult values by day 25 after birth. Medial preoptic and anterior mediobasal hypothalamic catecholamines exhibited a rather unique pattern. Concentrations and turnover rates were low in 15-day-old animals and increased between days 20 and 30 to very high values. Such high values were never observed in adult diestrous animals. The same pattern was also observed in the posterior mediobasal hypothalamus for NE concentrations and turnover rates whereas the respective values for DA showed relatively large fluctuations. On the basis of catecholamine measurements 30 and 90 min after blockade of tyrosine hydroxylase an attempt was also made to differentiate turnover rates of the functional and of the storage pool. Serum LH levels in the 15-day-old animals showed large fluctuations. FSH levels were high and prolactin levels were low. At the time of increased preoptic and hypothalamic NE and DA turnover rates, serum prolactin levels were also high whereas serum LH levels were lowest between days 20 and 30 and then slightly increased. Serum FSH levels were uniformly low. The possibility is discussed that high NE turnover may stimulate pituitary LH and prolactin release by hypothalamic mechanisms. Hihgh serum prolactin levels may stimulate DA turnover which is inhibitory to pituitary LH release, thus counteracting the stimulatory effect of NE on LH-RH release. The dopaminergic inhibition of LH may be relieved at the time of puberty partially because the DA receptors become desensitized to the action of DA.

Changes in catecholamine turnover in the anterior part of the mediobasal hypothalamus and the medial preoptic area in response to hyperprolactinemia in ovariectomized rats.

High serum prolactin levels in ovariectomized rats were induced by transplantation of additional pituitaries under the kidney capsule. Such high prolactin levels reduced serum LH but not FSH levels 3 days after pituitary transplantion. LH and FSH values were at a control levels at day 15 and above these levels at day 24, although prolactin values were still high. Dopamine (DA) and norepinephrine (NE) turnover in the anterior part of the mediobasal hypothalamus (AMBH) and in the medial preoptic area (MPO) was measured by following the decrease in NE or DA content after synthesis blockage with a-methyl-p-tyrosine. The content was measured using a radioenzymatic assay. DA turnover in the AMBH was significantly increased 3, 15 and 24 days after induction of hyperprolactinemia. Although NE concentration in the AMBH was also increased at these times an increased NE turnover was statistically significant only at day 24. DA as well as NE turnover rates in the MPO were reduced by day 3 after pituitary transplantation and at control values at day 15 and 24. It is concluded that the increased DA turnover in the AMBH depresses pituitary LH release probably by inhibiting hypothalamic LH-RH secretion. This inhibition is counteracted by the somewhat slower increase of NE turnover resulting in normalization of LH levels. The increased gonadotropin levels after long-lasting hyperprolactinemia may be due to desensitization of the DA receptor, which was reported earlier. The reduced NE turnover in the MPO 3 days after induction of hyperprolactinemia may be an additional factor in reducing pituitary LH release acting at the level of LH-RH-producing perikarya.

Involvement of catecholamines in eliciting LH peaks in 15-day-old female rats: effects of treatment with prolactin.

In the serum of 15-day-old female rats, treated either with alpha-methyl-p-tyrosine (alpha-MPT) or saline, serum LH levels were determined. From the brains of all animals the medial preoptic areas (MPO) and the anterior and posterior mediobasal hypothalami (AMBH and PMBH) were punched out and stored frozen in perchloric acid. In the punches of those animals with very high or basal LH levels catecholamine concentrations were measured radioenzymatically and on the basis of the degree of catecholamine depletion after alpha-MPT the turnover rates of dopamine (DA) and norepinephrine (NE) were calculated. NE turnover in the MPO was significantly higher in the animals with high LH levels as compared to those with low LH values. In the PMBH an inverse correlation between LH levels and NE turnover rates could be demonstrated. NE turnover in the AMBH, and DA turnover in all 3 structures did not correlate with serum LH levels. Serum FSH and prolactin levels were higher in the animals with high LH levels. These changes are similar to those observed in adult proestrous rats and it is concluded that the LH peaks in 15-day-old animals reflect a positive feedback action of estradiol. In a second experiment prolactin was administered to determine if it could prevent the occurrence of these LH peaks. Ovine prolactin (2 X 0.5 micrograms/g body weight) injected from day 8--15 suppressed serum LH levels to almost undetectable values. Catecholamine turnover measurements in these animals revealed increased DA turnover in the MPO, AMBH and PMBH, whereas NE turnover was not affected. These results indicate that DA turnover of the incertohypothalamic and of the tuberoinfundibular DA neurons is accelerated by prolactin and that an increased DA turnover in one of these systems is responsible for inhibition of LH release.

Evaluation and characterization of forage Sorghum as feedstock for fermentable sugar production.

Sorghum is a tropical grass grown primarily in semiarid and drier parts of the world, especially areas too dry for corn. Sorghum production also leaves about 58 million tons of by-products composed mainly of cellulose, hemicellulose, and lignin. The low lignin content of some forage sorghums such as brown midrib makes them more digestible for ethanol production. Successful use of biomass for biofuel production depends on not only pretreatment methods and efficient processing conditions but also physical and chemical properties of the biomass. In this study, four varieties of forage sorghum (stems and leaves) were characterized and evaluated as feedstock for fermentable sugar production. Fourier transform infrared spectroscopy and X-ray diffraction were used to determine changes in structure and chemical composition of forage sorghum before and after pretreatment and the enzymatic hydrolysis process. Forage sorghums with a low syringyl/guaiacyl ratio in their lignin structure were easy to hydrolyze after pretreatment despite the initial lignin content. Enzymatic hydrolysis was also more effective for forage sorghums with a low crystallinity index and easily transformed crystalline cellulose to amorphous cellulose, despite initial cellulose content. Up to 72% hexose yield and 94% pentose yield were obtained using modified steam explosion with 2% sulfuric acid at 140 degrees C for 30 min and enzymatic hydrolysis with cellulase (15 filter per unit (FPU)/g cellulose) and beta-glucosidase (50 cellobiose units (CBU)/g cellulose).

[Submerse bacterial plant tumor on roots]. / Submerser Pflanzenkrebs an Wurzeln.

Spontaneous infections seem to indicate that not only the crown of herbeous plants is disposed to generate tumors. Under substrate conditions such as those normally found at the crown, infections caused by Agrobacterium tumefaciens will yield tumors in the entire area of the roots.Roots of Helianthus annuus and Solanum lycopersicum grown in nutritive medium are disposed to tumor when given appropriate conditions of culture and inoculation. Tissue culture, the most widely applied method for the analysis of bacterial plant tumor, has a disadvantage in the correlation with the whole organism is lacking. This disadvantage is avoided when submerse cultures on intact roots are used, and the methodical advantages involved in using tissue cultures are preserved.When two unfavorable factors such as lack of oxygen and suboptimal temperature come together, a slowing down of tumoral induction results in hydroponic culture. Tumors already induced will grow normally. When the induction temperature is optimal (for Helianthus annuus 26°C) lack of oxygen will not produce inhibition of induction. Lack of oxygen will, however, unfavorably effect the total development of the roots in any case.Proportional dependence between the growth of the roots and that of the tumor does not exist. Tumors of medium size correspond to a maximal weight of roots. This paper discusses the relationship between the above mentioned phenomena and growth hormones.In the tests involving lack of oxygen epinasty could be observed.

Processivity of the DNA polymerase alpha-primase complex from calf thymus.

The processivity of the DNA polymerase alpha-primase complex from calf thymus was analyzed under various conditions. When multi-RNA-primed M13 DNA was used as the substrate, the DNA polymerase alpha-primase complex was found to incorporate 19 +/- 3 nucleotides per primer binding event. This result was confirmed by product analysis on sequencing gels following DNA synthesis on poly(dT) X (rA)10. The processivity depends strongly on the assay conditions but does not correlate with enzymic activity. Lowering the concentration of Mg2+ ions to less than 2 mM increases the processivity to 60. Replacing Mg2+ by 0.2 mM Mn2+ results in 90 nucleotides being incorporated per primer binding event. Neither the presence of ATP nor the addition of noncognate deoxynucleotide triphosphates affects the processivity of the DNA polymerase alpha-primase complex. Lower processivity was induced by lowering the reaction temperature, by adding spermine, spermidine, or putrescine, in the presence of the antibiotics novobiocin and ciprofloxacin, by adding Escherichia coli single-stranded DNA binding protein, or by adding calf thymus topoisomerase II and RNase H. Three single-stranded DNA binding proteins from calf thymus, including unwinding protein 1, do not affect processivity to any significant extent. Freshly prepared DNA polymerase alpha-primase complex exhibits in addition to its processivity of 20 further discrete processivities of about 55, 90, and 105. This result suggest that further subunits of the polymerase alpha-primase complex are necessary to reconstitute the holoenzyme form of the eukaryotic replicase.

The role of monoamines in female puberty.

The estradiol positive feedback mechanism appears to become mature between days 10 and 20 after birth. Rising serum prolactin levels between day 20 after birth and puberty are correlated with high hypothalamic norepinephrine turnover. High prolactin levels stimulate hypothalamic dopamine (DA) turnover, which may actively inhibit hypothalamic luteinizing hormone-releasing hormone (LHRH) release. Hypothalamic DNA receptor sensitivity is high in 10- to 20-day-old rats and gradually decreases between day 20 after birth and puberty. The reason for this desensitization may be the high hypothalamic DA turnover. This may result in a less strong inhibition of LHRH release allowing the positive feedback action of estradiol to elicit the first preovulatory luteinizing hormone (LH) surge initiating puberty.