Measurement of Laser-Plasma Electron Density with a Soft X-ray Laser Deflectometer.

A soft x-ray laser (wavelength lambda = 15.5 nanometers) was used to create a moiré deflectogram of a high-density, laser-produced plasma. The use of deflectometry at this short wavelength permits measurement of the density spatial profile in a long-scalelength (3 millimeters), high-density plasma. A peak density of 3.2 x 10(21) per cubic centimeter was recorded.

Spontaneous formation of germinal centers in autoimmune mice.

The mechanisms of autoantibody production are not well understood. Germinal centers (GC) may be important sites of immune disregulation in autoimmune diseases. In this study, we document the presence of spontaneous GC formation in the spleens of several autoimmune mouse strains that spontaneously develop autoimmune Type I diabetes and a lupus-like disease. In contrast, mouse strains that do not develop lupus did not exhibit spontaneous formation of GC. In all of the autoimmune strains studied, GC were present at 1-2 months of age, a time that closely parallels the appearance of autoantibodies. Like the GC that develop after purposeful immunization, GC in autoimmune mice contained B220(+), PNA(+), and GL-7(+) B cells, and FDC-M1(+) follicular dendritic cells. In addition, spontaneously formed GC in autoimmunity and those caused by immunization were abrogated in a similar way by a short-term treatment with anti-CD40 ligand antibody. These data indicate that spontaneously forming GC in autoimmunity are similar to those appearing after purposeful immunization.

Beta-2 adrenoceptor blocking activity of penbutolol and propranolol at very low doses.

The relative potency of penbutolol, a new beta adrenergic receptor blocking agent, was compared with propranolol by a four-point assay on healthy male subjects. A dose-response relationship to intravenous doses of propranolol in the microgram range was obtained during a steady state of infusion of epinephrine. Two subjects underwent the entire assay according to the Latin square design and four others each underwent one schedule of design on different days. The potency of penbutolol thus assessed was consistent with reported results. This study emphasizes the importance of intersubject variation and differential receptor sensitivity in individuals. The use of epinephrine as a beta receptor adrenergic agonist for evaluation of selective beta adrenergic receptor blocking activities is discussed.

Molecular cloning of FKHRL1P2, a member of the developmentally regulated fork head domain transcription factor family.

Here we report the expression of a fork head domain protein in human T helper cells. We cloned and characterized a fork head cDNA from human T helper cell mRNA using differential display RT-PCR. The cDNA contains a 546-nucleotide (nt) open reading frame (ORF) that codes for the carboxyl-terminal 180 amino acids (aa) of the recently identified fkhrl1 gene. This ORF does not contain the characteristic DNA-binding domain found in members of the forkhead protein family. In-vitro transcription/translation of this cDNA expressed a protein of approximately 20 kDa. We have generated antibodies that specifically immunoprecipitated the in-vitro-translated 20-kDa protein. This antibody also recognizes in human T lymphocytes a 70-kDa protein corresponding in size to that predicted for the fkhrl1 gene product. The mRNA levels for fkhrl1 is elevated in T helper-induced lymphocytes in comparison to PHA-stimulated T lymphocytes. Further characterization of FKHRL1 and its related family members should shed light on the transcriptional mechanisms of this fork head gene subfamily and their role in T helper cell differentiation and regulation of cell growth.

Prolactin recruits STAT1, STAT3 and STAT5 independent of conserved receptor tyrosines TYR402, TYR479, TYR515 and TYR580.

The present study of prolactin (PRL) receptor-mediated recruitment of signal transducers and activators of transcription (STATs) demonstrates that PRL activates STAT3, in addition to STAT1 and STAT5 as previously reported, and that STAT1, STAT3 and STAT5 are mediators of PRL effects in cells whether of lymphoid, myeloid or mammary epithelial origin. Furthermore, receptor mutants M240 and T280 that do not mediate PRL-induced JAK2 activation and cell proliferation, are also unable to mediate STAT activation, supporting the proposed model of JAK2 as the initial effector protein used by PRL receptors. On the other hand, tyrosine phosphorylation analysis and electrophoretic mobility shift assays showed that receptor mutant G328, which lacks four of the five conserved cytoplasmic tyrosine residues of PRL receptors, retained the ability to activate JAK2 and STAT1, STAT3 and STAT5. These results support the notion that phosphotyrosyl residues other than those of the receptor, i.e., JAK2, are involved in recruiting STAT proteins to the activated PRL receptor complex.

Dental OCT.

We present here the first in vivo optical coherence tomography (OCT) images of human dental tissue. A novel dental optical coherence tomography system has been developed. This system incorporates the interferometer sample arm and transverse scanning optics into a handpiece that can be used intraorally to image human dental tissues. The average imaging depth of this system varied from 3 mm in hard tissues to 1.5 mm in soft tissues. We discuss the application of this imaging system for dentistry and illustrate the potential of our dental OCT system for diagnosis of periodontal disease, detection of caries, and evaluation of dental restorations.

A biochanin-enriched isoflavone from red clover lowers LDL cholesterol in men.

OBJECTIVE: To determine whether the two major isoflavones in red clover differ in their effect on low-density lipoprotein cholesterol (LDL-C). DESIGN: A randomised, placebo-controlled, double-blind trial; two parallel groups taking one of the two isoflavones within which treatment and placebo were administered in a crossover design. SETTING: Free-living volunteers. SUBJECTS: A total of 46 middle-aged men and 34 postmenopausal women. INTERVENTION: Two mixtures of red clover isoflavones enriched in either biochanin (n=40) or formononetin (n=40) were compared. Placebo and active treatment (40 mg/day) were administered for 6 weeks each in a crossover design within the two parallel groups. MAIN OUTCOME MEASURES: Plasma lipids were measured twice at the end of each period. RESULTS: Baseline LDL-C concentrations did not differ significantly between men (n=46) and women (n=34), nor between those randomised to biochanin or formononetin. Interaction between time and treatments, biochanin, formononetin and corresponding placebos (two-way ANOVA) on LDL-C showed a significant effect of biochanin treatment alone. The biochanin effect was confined to men; median LDL-C was 3.61 (3.05-4.14) mmol/l with biochanin and 3.99 (3.16-4.29) mmol/l with the corresponding placebo (RM ANOVA with Dunnett's adjustment P<0.05). The difference between placebo and biochanin effects on LDL-C was 9.5%. No other lipid was affected and women failed to respond significantly to treatment. CONCLUSION: Isolated isoflavones from red clover enriched in biochanin (genistein precursor) but not in formononetin (daidzein precursor), lowered LDL-C in men. This may partly explain the previous failure to demonstrate cholesterol-lowering effects with mixed isoflavones studied predominantly in women. SPONSORSHIP: Novogen Ltd, North Ryde NSW, Australia, provided partial support including provision of tablets and outside monitoring.

Mechanisms of cytokine signal transduction: IL-2, IL-4 and prolactin as hematopoietin receptor models.

Cytokines, hormones and hematopoietic growth factors transduce biological signals across the cell membrane via a highly conserved family of single membrane-spanning receptors. The intracellular signal transducing machinery responsible for mediating these responses has remained largely unknown. However, recent identification of a homologous class of tyrosine kinases, Janus Kinases (JAKs), and a related family of transcription factors, signal transducers and activators of transcription (STATs), has shed new light on the molecular mechanisms responsible for mediating hematopoietin signaling and immune response. Current research efforts within the field of cytokine signaling have now shifted to understanding how these molecules are activated by hematopoietic receptors, positively and negatively regulated by kinases and phosphatases, and how they impact on gene transcription to ultimately coordinate cell homeostasis, proliferation and differentiation. This article will review some of our results identifying the involvement of JAKs, STATs, and secondary effector molecules activated following engagement of hematopoietic receptors for IL-2, IL-4, and prolactin. Here, we provide evidence for the ingenious ability of cytokine receptors to selectively recruit and activate these proteins among a repertoire of possible alternative biochemical messengers as a means to affect unique and general cell responses.

A tobacco cDNA reveals two different transcription patterns in vegetative and reproductive organs.

In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8) showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5' sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt) long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries). Plants submitted to stress (wounding, virus infection and ethylene treatment) presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger.

[Cell-based therapy to treat stress urinary incontinence: which cell type at what cost?]. / Zellbasierte Therapie der Belastungsinkontinenz: Welche Zelle zu welchen Kosten?

In Germany, 6-8 million woman and men suffer urinary incontinence, which represents 12.5 % of the population. It is estimated that by the middle of this century, it will increase to almost 30 %. The primary reason will be primarily related to the aging population but also to patient awareness and seeking a solution. In addition to the cost which is covered by the health insurance, the patient will spend more than half a billion euro/year out-of-pocket, not to mention the social stigma associated with urinary incontinence. The current common treatment options are symptomatic but do not restore functionality. One option might be tissue engineering or stem cell therapy. This article describes the likelihood that this therapy will change the approach in treating stress urinary incontinence. Boundaries and legal aspects are highlighted as well as approximated cost. These treatment costs might be currently higher than the standard treatment options, but the investment to reduce these costs are paid indirectly by society.

Equine infectious anemia virus tat: insights into the structure, function, and evolution of lentivirus trans-activator proteins.

Equine infectious anemia virus (EIAV) contains a tat gene which is closely related to the trans-activator genes of the human and simian immunodeficiency viruses. Nucleotide sequence analysis of EIAV cDNA clones revealed that the tat mRNA is composed of three exons; the first two encode Tat and the third may encode a Rev protein. Interestingly, EIAV Tat translation is initiated at a non-AUG codon in exon 1 of the mRNA, perhaps allowing an additional level of gene regulation. The deduced amino acid sequence of EIAV tat, combined with functional analyses of tat cDNAs in transfected cells, has provided some unique insights into the domain structure of Tat. EIAV Tat has a C-terminal basic domain and a highly conserved 16-amino-acid core domain, but not the cysteine-rich region, that are present in the primate immunodeficiency virus Tat proteins. Thus, EIAV encodes a relatively simple version of this kind of trans activator.

Structure and expression of the equine infectious anemia virus transcriptional trans-activator (tat).

Equine infectious anemia virus (EIAV) encodes a tat gene which is closely related to the trans-activators encoded by the human and simian immunodeficiency viruses. Nucleotide sequence analysis of EIAV cDNA clones revealed that the tat message is composed of three exons; the first two encode tat and the third may encode rev.. Interestingly, EIAV tat translation is initiated at a non-AUG codon in the first exon of the message, perhaps allowing an additional level of gene regulation. The deduced amino acid sequence of EIAV tat, combined with functional analyses of tat cDNAs in transfected cells, have provided some unique insights into the domain structure of this protein. EIAV Tat has a C-terminal basic domain, a highly conserved 16 amino acid core domain, but not the cysteine-rich region, that is present in the primate immunodeficiency virus Tat proteins. Thus EIAV encodes a relatively simple version of this kind of trans-activator.

Cryopreservation of reduced cytochrome C for determination of N-formyl-methionyl-leucyl-phenylalanine-stimulated superoxide anion production in human whole blood.

Various methods are available for measuring the production of reactive oxygen species by phagocytes, but they are limited in their use by the need for their immediate application, cell isolation and of cell-activation by unphysiological stimuli. In addition, after measurement of reactive oxygen metabolites using oxidizing agents, the reduced compounds formed have to be determined during or immediately after their formation. In the present study, an improved cytochrome C assay was investigated which allowed measurements of superoxide anions in whole blood samples following activation of phagocytes by physiological stimuli such as the bacterial tripeptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). The fMLP-stimulated production of superoxide anion (O(2)(-)) showed a sigmoidal-shaped fMLP dose-response curve, and constant O(2)(-) production rates (nmol.1(-1)x10(6) granulocytes) could be determined reliably up to a blood granulocyte concentration of 1 x 10(4) x microl(-1). To allow the determination of reduced cytochrome C later after its formation, the effect of long-term storage at -20 degrees C on the stability of reduced cytochrome C was tested up to 16 weeks. The results obtained show that the determination of reduced cytochrome C in whole blood represents a simple and reliable method. Most importantly, O(2)(-)-reduced cytochrome C can be frozen and stored without any alterations, at least up to 2 weeks. Thus the method seems to be superior to other methods of detection, especially when the experimental conditions do not allow immediate spectrophotometry (e.g. mountain medicine, space medicine). Under such conditions the present assay would allow reliable measurement of reduced cytochrome C, even after weeks of cryopreservation.

Immunocompromised tumor-bearing mice show a selective loss of STAT5a/b expression in T and B lymphocytes.

Impaired immune responses are frequently observed in tumor-bearing hosts during progression of tumor growth, but the molecular basis of these functional defects remains unclear. To investigate tumor-induced immunosuppression, we first established that lymphocytes from mice bearing s.c. mammary adenocarcinoma (TS/A) tumors were severely impaired in their ability to generate cellular and humoral Ag-specific responses. Lymphocytes from these mice were then screened for abnormalities in the expression of signal transducing proteins known to be involved in the regulation of cellular immunity. Interestingly, purified T and B cells isolated from immunocompromised tumor-bearing mice displayed a marked decrease in the transcription activators STAT5a and -b at the protein level and to a lesser extent at the mRNA level. By contrast, no change in the expression of STAT1, -3, and -6 or of the TCR itself were detected. The correlation in the loss of T and B cell function with the selective decrease in STAT5a/b expression suggests that regulation of the STAT5 signaling pathway may provide a molecular mechanism for modulating the immune system.

Growth signaling and JAK2 association mediated by membrane-proximal cytoplasmic regions of prolactin receptors.

Prolactin (PRL) has recently been demonstrated to induce tyrosine phosphorylation and activation of the cytoplasmic tyrosine kinase JAK2 in PRL-dependent Nb2 lymphoma cells. The present study represents an initial effort to identify cytoplasmic regions of the PRL receptor (PRLR) that are critical to growth signal generation and JAK2 activation. Variably truncated rat PRLRs were stably expressed in the murine 32D cell line. PRL-induced proliferation was mediated by the full-length receptor and the mutant G328, which contains the membrane-proximal Homology Boxes 1 and 2 characteristic of hematopoietin receptors. In contrast, mutant receptors lacking either Box 2 or both Boxes 1 and 2 were not capable of transmitting a growth signal. The mitogenic capacity of the PRLR variants correlated with JAK2 association and activation, as well as with induction of mRNA levels for the growth-related gene ornithine decarboxylase. We conclude that mitogenic competence of rat PRLRs resides within the first 94 amino acids of the cytoplasmic domain. The data suggest that Homology Box 1/Box 2 region is critical to JAK2 phosphorylation and association with the PRLR. These findings will serve as a basis for more detailed molecular characterization of PRLR domains essential for JAK2 interaction and growth signal generation.