On the evocability of a positive oestrogen feedback action on LH secretion in transsexual men and women.

In transsexual men with homosexual behaviour and intact testicular function, as well as in homosexual men with normal gender identity, following a negative oestrogen feedback effect a delayed positive oestrogen feedback action on LH secretion was evoked. By contrast, in transsexual men with hypo- or asexuality and intact testes or hypergonadotrophic hypo- or agonadism, as well as in heterosexual men with normal gender identity, a negative oestrogen feedback effect was not followed by a positive feedback action on LH release. In transsexual women with homosexual behaviour and oligo- and/or hypomenorrhoea, only a weak or at best moderate positive oestrogen feedback action on LH release was evocable, similarly as in castrated and oestrogen-primed heterosexual men. By contrast, in a transsexual woman with bisexual behaviour and eumenorrhoea, a strong positive oestrogen feedback action on LH secretion was evocable, as well as in heterosexual women with normal gender identity.     

Regulation of luminal membrane water permeability by water flow in toad urinary bladder

Intramembranous particle aggregates (presumed sites for water flow) which appear in the luminal membrane consequent to ADH treatment are derived from cytoplasmic membrane structures (now termed "aggrephores") which fuse with the luminal membrane. We have previously shown that bladders stimulated in the absence of an osmotic gradient have about twice as many aggregates and about three times as many sites of aggrephore fusion as bladders stimulated with ADH in the presence of a 175 milliosmolal gradient. The present studies show that the frequency of fused aggrephores and luminal membrane aggregates can be modified as a consequence of alterations in transmembrane water flow initiated by changing the transbladder osmotic gradient during hormone stimulation. Bladders treated with ADH for 1 hr without a gradient and then for 1 hr with a gradient had approximately 1/3 as many aggregates and fusion sites as paired bladders treated for 2 hr without a gradient. Conversely, bladders treated with ADH for 1 hr with a gradient and then for 1 hr without a gradient had approximately 2x as many aggregates and fusion sites as bladders treated for 2 hr with a gradient. In other experiments we demonstrate that the time course of hormone washout is greatly accelerated if carried out in the presence of an osmotic gradient. In paired bladders that were first stimulated with ADH for 30 min in the absence of a gradient, aggregates and fusion sites as well as osmotic water permeability determined in fixed bladders, persisted at near maximum levels for 15 min of washout in the absence of a gradient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recovery and purification of aprotinin from industrial insulin-processing effluent by immobilized chymotrypsin and negative IMAC chromatographies

Aprotinin, a bovine protease inhibitor currently also produced in recombinant bacteria, yeast, and corn, has valuable applications as a human therapeutic and in tissue culture. The objective of this work was to develop the basis of a large-scale aprotinin purification process centered on immobilized metal ion affinity chromatography (IMAC). This technique uses ligands—metal ions—of a lower cost and higher stability than those traditionally used in affinity chromatography. Since aprotinin does not interact with IMAC ligands, collection is from the nonretained fractions (negative chromatography). Stirred-tank batch IMAC adsorption experiments indicated that one-step aprotinin purification could not be successful. Immobilized chymotrypsin chromatography was then used as a prepurification step, yielding a suitable feed for IMAC (with purification factors as high as 476). IMAC column fed with these prepurified materials produced purified aprotinin in the nonretained fractions with purification factors as high as 952.     

Isolation and identification of hydroxyl-platelet-activating factor from natural sources

Platelet-activating factor (PAF), a potent inflammatory mediator that has previously been detected in elevated levels in inflamed gingival tissues, in gingival crevicular fluid (GCF) and in saliva, is implicated in periodontal disease. The biologically active phospholipid detected in gingival crevicular fluid is a hydroxyl-PAF analogue. In a preliminary study this bioactive molecule was detected for the first time in human blood derived from volunteers with chronic periodontitis as well as from periodontally healthy volunteers. Compounds isolated from natural sources as well as synthetic ones have been reported as biologically active lipids with physiological importance based on the fact that they induce platelet aggregation with EC50 values ranging from 100 to 0.01 microM through interaction with G-protein-coupled receptors like the PAF receptor, leading to altered signal transduction. In this study, the existence of hydroxyl-PAF analogue in human blood was further studied as well as its distribution in plasma and in blood components. The existence of hydroxyl-PAF analogue was also investigated in samples from rabbit blood hen's egg yolk. The hydroxyl-PAF analogue was purified by high-performance liquid chromatography, detected by biological assays and identified by electrospray MS analysis. Quantitative determination of PAF and hydroxyl-PAF analogue (expressed as PAF-like activity) showed a statistically significant increase in the ratio of plasma hydroxyl-PAF analogue levels to plasma PAF levels in volunteers with periodontitis. Moreover, hydroxyl-PAF analogue was also detected in rabbit blood and hen's egg yolk samples. These data support that this bioactive lipid may play a role in oral inflammation and suggest PAF as a member of a lipid molecule family with different structures and from different sources which share the same or similar biological activities, apparently with different physiological roles in human and animals.     

Hepcidin assay in serum by SELDI-TOF-MS and other approaches

Hepcidin, a liver peptide hormone, is the central regulator of iron homeostasis. Hepcidin synthesis is modulated by iron stores, so that iron repletion increases its levels to prevent pathological overload, while iron deficiency strongly inhibits hepcidin to allow an increase in iron absorption from duodenal cells. The emerging pivotal role of hepcidin in iron homeostasis, along with its important links with basic pathways like inflammation, makes the availability of an accurate hepcidin assay as a potentially powerful investigative tool to improve our understanding as well as our diagnostic/prognostic capabilities in many human diseases. There has been a great interest worldwide in developing a reliable and widely applicable assay of the hormone in biological fluids. Being optimal for low-molecular-weight biomarkers, SELDI-TOF-MS has emerged as a valid tool for hepcidin assay. Here we review recent results obtained with this technique, as well as with other Mass Spectrometry-based and immunological methods.     

A trans-acting factor, isolated by the three-hybrid system, that influences alternative splicing of the amyloid precursor protein minigene.

Two clones were isolated in a three-hybrid screen of a rat fetal brain P5 cDNA library with an intronic splicing enhancer of the amyloid precursor protein (APP) gene as RNA bait. These clones represent the rat homologues of the previously described genes CUG-binding protein (CUG-BP) and Siah-binding protein (Siah-BP). Both interact in a sequence-specific manner with the RNA bait used for library screening as well as with the CUG repeat. In contrast, no interactions were observed in the three-hybrid assay with other baits tested. In two-hybrid assays, Siah-BP interacts with U2AF65 as well as with itself. EWS, an RGG-type RNA-binding protein associated with Ewing sarcoma, was identified as an interacting partner for the CUG-BP homologue in a two-hybrid assay for protein-protein interactions performed with various factors involved in RNA metabolism. Splicing assays performed by RT-PCR from cells cotransfected with certain cDNAs and an APP minigene, used as a reporter, indicate exclusion of exon 8 if the CUG-BP homologue is present. We conclude that clone AF169013 and its counterpart in human CUG-BP could be the trans-acting factors that interact with the splicing enhancer downstream of exon 8, and in this way influence alternative splicing of the APP minigene.     

Circular dichroism of polynucleotides: dimers as a function of conformation

Working within the restrictions of a model, we have calculated the circular dichroism of the dinucleoside phosphates ApA, CpC, and CpA for various conformations. Comparing the calculated curves with those measured in aqueous solution we find agreement for (1) ApA as a right-handed helix with both bases either as in B-form DNA, or else rotated 180° around the glycosidic bond, (2) CpC as the right-handed conformation with both bases as in DNA, (3) ApC as either the right-handed conformation with both bases as in DNA, or else as a left-handed helix with both bases rotated 180°, and (4) CpA as either a left-handed helix with both bases in a left-handed DNA, or else in the right-handed conformation with both bases rotated 180°. In addition, we have investigated circular dichroism as a measure of unstacking. We find that opening the bases to a 90° total angle (base planes perpendicular) reduces the intensity of the calculated bands to 20% of their original value. Further, we find that allowing the sliding of one base past the other does not lead to a temperature dependence consistent with experiment.     

Dipeptidyl aspartyl fluoromethylketones as potent caspase inhibitors: SAR of the N-protecting group

This article describes the synthesis and biological evaluation of a group of N-protected Val-Asp-fmk as caspase inhibitors. The protecting group was found to contribute to caspase-3 inhibiting activity, and compounds with a large group such as Cbz are more active than compounds with a small group such as Ac. Compounds with more hydrophobic protecting groups were found to be more active in cell apoptosis protection assays, probably due to increased cell permeability. MX1122, 2,4-di-Cl-Cbz-Val-Asp-fmk, is identified as a potent broad-spectrum caspase inhibitor and is selective for caspases versus other proteases, with good activity in the cell apoptosis protection assays as well as good efficacy in the mouse liver apoptosis model.     

Protein kinase D: an intracellular traffic regulator on the move

Recent research has identified protein kinase D (PKD, also called PKCmu) as a serine/threonine kinase with potentially important roles in growth factor signaling as well as in stress-induced signaling. Moreover, PKD has emerged as an important regulator of plasma membrane enzymes and receptors, in some cases mediating cross-talk between different signaling systems. The recent discovery of two additional kinases belonging to the PKD family and the plethora of proteins that interact with PKD point to a multifaceted regulation and a multifunctional role for these enzymes, with functions in processes as diverse as cell proliferation, apoptosis, immune cell regulation, tumor cell invasion and regulation of Golgi vesicle fission.     

Counselling the parents of handicapped children.

The parents of handicapped children have to adjust to a wide variety of emotional and psychological problems when first confronted with the failure of their reproductive expectations. Counselling is a formal procedure or transaction in which both counsellor and parents aim to find a mutually acceptable plan of adjustment. Parents may need support to cope with their own personal inadequacies as well as with their own personal inadequacies as well as with the needs of the child. Counselling should be a continuous process, in which the parents can learn to accept the child as a different rather than a lesser person.     

Enhancement of phase separation using a drop coalescer in an aqueous two-phase system

The effect of a drop coalescer on phase separation in a PEG/salt aqueous two-phase system (ATPS) in the absence and presence of protein has been investigated. Raschig rings of ceramic, PTFE and glass were used as a drop coalescer in order to separate the mixture into two phases. Among the three materials PTFE is the most effective in coalescing the dispersed drops, with the throughput with PTFE being twice that without the coalescer. Random packing gives good results for phase separation. Two types of fiber mesh coated with PTFE were also used as drop coalescers, one in a spirally folded form and the other in a three-dimensional lattice-form. Throughput in the PEG/salt system with the three-dimensional lattice-form is 1.2 times as high as that with the spirally folded form. Throughput with the coalescer formed by compiling PTFE Raschig rings and fiber mesh in lattice form is 1.6 and 1.2 times as high as the case of separate use of the fiber mesh and the PTFE Raschig rings, respectively. The hydrophobic surface of PTFE in the compiled coalescer has no significant effect on the recovery fraction of the protein in ATPS.     

一种检测kras基因突变的新型TB-ARMS qPCR方法的建立

在荧光定量PCR基础上建立一种简单有效并且高度灵敏的TB-ARMSkras基因突变检测方法,并对其检测性能进行评估,探讨其临床应用价值。针对kras基因8种常见的点突变类型,通过设计并优化突变特异性引物、野生型特异性封闭引物并综合应用突变富集扩增反应条件等多种手段,提高点突变检测的灵敏度和特异性,采用已知野生型基因组样品和构建的突变质粒作为标准品,进行方法学评价;通过对临床样本的检测及与现有商品化试剂盒的比较进行性能验证;通过对术前血浆和配对组织样品的对比检测,评估方法是否适用于血液样本的检测。建立了TB-ARMS kras突变检测的新方法,能检测的最低突变率可达到0.01%。通过综合采用野生型特异性封闭引物和突变富集扩增条件等方法证明了其0.01%的突变检测灵敏度。检测准确性优于现有商品化试剂盒,血浆DNA TB-ARMS qPCR检测结果与配对组织DNA测序结果相符合。因此,TB-ARMS kras基因突变检测方法具有广泛的临床应用价值,既适用于临床组织样品的检测,也可应用于液体活检。     

Affect-modulated startle: interactive influence of catechol-O-methyltransferase Val158Met genotype and childhood trauma

The etiology of emotion-related disorders such as anxiety or affective disorders is considered to be complex with an interaction of biological and environmental factors. Particular evidence has accumulated for alterations in the dopaminergic and noradrenergic system--partly conferred by catechol-O-methyltransferase (COMT) gene variation--for the adenosinergic system as well as for early life trauma to constitute risk factors for those conditions. Applying a multi-level approach, in a sample of 95 healthy adults, we investigated effects of the functional COMT Val158Met polymorphism, caffeine as an adenosine A2A receptor antagonist (300 mg in a placebo-controlled intervention design) and childhood maltreatment (CTQ) as well as their interaction on the affect-modulated startle response as a neurobiologically founded defensive reflex potentially related to fear- and distress-related disorders. COMT val/val genotype significantly increased startle magnitude in response to unpleasant stimuli, while met/met homozygotes showed a blunted startle response to aversive pictures. Furthermore, significant gene-environment interaction of COMT Val158Met genotype with CTQ was discerned with more maltreatment being associated with higher startle potentiation in val/val subjects but not in met carriers. No main effect of or interaction effects with caffeine were observed. Results indicate a main as well as a GxE effect of the COMT Val158Met variant and childhood maltreatment on the affect-modulated startle reflex, supporting a complex pathogenetic model of the affect-modulated startle reflex as a basic neurobiological defensive reflex potentially related to anxiety and affective disorders.     

PIKA provides an adjuvant effect to induce strong mucosal and systemic humoral immunity against SARS-CoV

Severe Acute Respiratory Syndrome (SARS) is a deadly infectious disease caused by SARS Coronavirus (SARS-CoV). Inactivated SARS-CoV has been explored as a vaccine against SARS-CoV. However, safe and potent adjuvants, especially with more efficient and economical needle-free vaccination are always needed more urgently in a pandemic. The development of a safe and effective mucosal adjuvant and vaccine for prevention of emergent infectious diseases such as SARS will be an important advancement. PIKA, a stabilized derivative of Poly (I:C), was previously reported to be safe and potent as adjuvant in mouse models. In the present study, we demonstrated that the intraperitoneal and intranasal co-administration of inactivated SARS-CoV vaccine together with this improved Poly (I:C) derivative induced strong anti-SARS-CoV mucosal and systemic humoral immune responses with neutralizing activity against pseudotyped virus. Although intraperitoneal immunization of inactivated SARS-CoV vaccine alone could induce a certain level of neutralizing activity in serum as well as in mucosal sites, co-administration of inactivated SARS-CoV vaccine with PIKA as adjuvant could induce a much higher neutralizing activity. When intranasal immunization was used, PIKA was obligatorily for inducing neutralizing activity in serum as well as in mucosal sites and was correlated with both mucosal IgA and mucosal IgG response. Overall, PIKA could be a good mucosal adjuvant candidate for inactivated SARS-CoV vaccine for use in possible future pandemic.     

Solvent influence on base stacking.

In this paper we present a detailed analysis of the base-stacking phenomenon in different solvents, using nanosecond molecular dynamics simulations. The investigation focuses on deoxyribo- and ribodinucleoside monophosphates in aqueous and organic solutions. Organic solvents with a low dielectric constant, such as chloroform, and solvents with intermediate dielectric constants, such as dimethyl sulfoxide and methanol, were analyzed. This was also done for water, which is highly polar and has a high dielectric constant. Structural parameters such as the sugar puckering and the base-versus-base orientations, as well as the energetics of the solute-solvent interactions, were examined in the different solvents. The obtained data demonstrate that base stacking is favored in the high dielectric aqueous solution, followed by methanol and dimethyl sulfoxide with intermediate dielectric constants, and chloroform, with a low dielectric constant.     

The nature of blast cells in myelodysplastic syndromes evolving to acute leukaemia

The blast cells from nine patients with an overt acute leukaemia following a previous myelodysplastic syndrome (MDS) are analyzed with a panel of monoclonal antibodies as well as by morphological and cytochemical criteria. By integrating the results obtained with these three approaches the leukaemia in 6 patients was assessed as myeloid-granulocytic and/or monocytic-, in two as mixed- megakaryoblastic/myeloid- and in one as lymphoid. A good correlation between morphology, cytochemistry and immunological markers was observed in 7 out of the 9 cases. In three cases a noteworthy percentage of J5+ cells was detected. The exceptional finding of lymphoid as well as megakaryocytic and myeloid transformations suggests that the target cell for these leukaemias could be a pluripotent stem cell.     

Validation of an ultrasensitive and specific immunofluorometric assay for mouse follicle-stimulating hormone

Sensitive and specific measurement of FSH is critical to research in reproductive biology, and the increasing availability of transgenic mouse models has created a need for a robust, sensitive, and specific mouse (m) FSH assay. The present study evaluated a time-resolved immunofluorometric assay (IFMA) for mFSH using monoclonal antibody to human (h) FSHbeta as a capture antibody and a biotinylated polyclonal antibody to rat alpha subunit as a detection probe, with signaling amplified by europium-labeled streptavidin. The mFSH IFMA lowered the detection limit 34-fold (5 vs. 170 pg/sample) compared with standard mFSH RIA. The mFSH IFMA demonstrated parallelism of response to dilutions of castrated mouse serum and rat FSH but no cross-reactivity with hFSH and mLH or hLH, whereas the RIA demonstrated nonparallel cross-reactivity with hFSH. The IFMA has a wide analytical range, with a good precision profile for within- and between-assay reproducibility. Because the IFMA is a sandwich-type assay with strict dimer-specificity by design, the lower readings and recovery obtained were compared with the RIA when both assays used a pituitary-purified mFSH assay standard that contained isolated or fragmented subunits as well as intact dimeric FSH. When used with mouse serum sample, the mFSH IFMA demonstrated the expected increases following orchidectomy as well as markedly enhanced sensitivity to very low levels of endogenous mFSH in gonadotropin-deficient mice. Furthermore, the IFMA measured mFSH with fidelity in both intact and orchidectomized male mice without any interference from transgenic hFSH. The greatly enhanced sensitivity, specificity, and technical convenience of this mFSH IFMA will allow wider application of FSH measurements to very small blood samples in immature and mature mice as well as transgenic models.     

Hexokinase activity as marker to assess time of harvest of foot-and-mouth disease virus in BHK21 Cl13 cell cultures

Hexokinase (B.C. 2.7.1.1) activity as a marker enzyme during FMD viral infection has been observed spectrophotometrically in a system coupled with glucose-6-phosphate dehydrogenase, in supernatants of BHK(21)Cl(13) suspension as well as anchored cell culture at a minimum of 10(4) infective virus particles/ml. Specific activity increased with virus concentration in culture supernatants and abruptly decreased with a fall in virus titer, as has been noted by TCID/50,146 S concentration, and enzyme-linked immunosorbent assay (ELISA) readings.