Gonadotrophin-releasing hormone agonist dose-dependency of pituitary desensitization during controlled ovarian hyperstimulation in IVF

The aim of this study was to find the minimal effective daily s.c. dose of the gonadotrophin-releasing hormone (GnRH) agonist, triptorelin acetate, that suppresses the GnRH-induced release of luteinizing hormone (LH) at time of human chorionic gonadotrophin (HCG) injection and thereby prevents spontaneous LH surges during in-vitro fertilization (IVF) stimulation cycles. Therefore, a double-blind, prospective and randomized titration study was performed. A total of 48 IVF patients were divided into four groups of 12 patients. Each group received a different dose of triptorelin acetate, namely 5, 15, 50 or 100 microg s.c. daily. Standard ovarian stimulation was carried out using urinary follicle stimulating hormone (FSH) preparations. A 500 microg GnRH test was performed 90 min before the HCG injection in order to measure the degree of pituitary desensitization. Spontaneous LH surges were not detected in any of the groups, although three patients in the 5 microg group had ovulated at the time of ovum retrieval. The pituitary LH response to the GnRH test at time of HCG, expressed as area under the curve (AUC), appeared to be dose-dependent. Thus, a daily s.c. dose of 100 microg triptorelin acetate appears to be too high, since adequate desensitization of the pituitary (i.e. no spontaneous LH surge) can be achieved with doses as low as 15 and 50 microg.      

B lymphocytes secreting IgG linked to latent transforming growth factor- beta prevent primary cytolytic T lymphocyte responses

B lymphocytes secreting IgG linked to latent transforming growth factor (TGF)-beta (IgG-TGF-beta) prevent cytolytic T lymphocyte (CTL) responses to unrelated antigens in mixed lymphocyte cultures (MLC) so long as resting resident macrophages and functional Fc receptors are present. This was shown using IgG-secreting plaque-forming cells (PFC) to sheep erythrocytes (SRBC) obtained from popliteal lymph nodes of mice injected repeatedly in foot pads with SRBC. Remarkably, as few as approximately 300 PFC prevented CTL responses of 5 x 10(5) normal syngeneic spleen cells in MLC. Supranatants of short-term cultures of PFC also prevented CTL responses, and suppression was prevented by eliminating or dissociating IgG and TGF-beta present in supranatants or by antibody against active TGF-beta. Furthermore, the latency- associated peptide of latent TGF-beta was detected in approximately 10% of foci of IgG captured from single PFC, indicating that at least some B lymphocytes secrete IgG-TGF-beta as a complex. Resting resident macrophages (which do not produce latent TGF-beta) and functional Fc receptors were required for suppression, consistent with idea that IgG- TGF-beta is taken up through Fc receptors for IgG and that active TGF- beta, cleaved from latent TGF-beta of the complex, is delivered directly to potentially responding CTL. If CTL responses in man are similarly regulated by B lymphocytes, then an ongoing B cell response in patients with chronic viral infections or bearing immunogenic cancers may prevent effective therapeutic vaccination.      

Cell membrane-associated MT1-MMP-dependent activation of pro-MMP-2 in A375 melanoma cells.

Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, can degrade extracellular matrix components under physiological conditions and during cancer invasion and metastasis. Among the MMPs, the 72 kDa type IV collagenase MMP-2 (gelatinase A) is activated in a membrane-associated manner by an activation complex composed of membrane type 1 matrix metalloproteinase (MT1-MMP), tissue inhibitor of matrixmetalloproteinase-2 (TIMP-2), and pro-MMP-2 in the presence of alphavbeta3 integrin receptor. The activation of pro-MMP-2 correlates with increased occurrence of metastases. Increased MMP-2 activity has been demonstrated in many human tumors. In the present communication, we studied cell surface-associated activation of MMP-2 (72 kDa collagenase type IV) in the moderately metastatic human melanoma cell line A375. RESULTS: Activation of purified 72 kDa collagenase type IV, pro-MMP-2 from cervical cancer tissue homogenate and from serum-free culture medium of HT1080 cells grown in presence of concanavalin A, by A375 cells, was shown by gelatin zymography. A375 cells activated only pro-MMP-2 from purified MMP-9/MMP-2 mixture indicating that the activation is specific for MMP-2. Activation of MMP-2 and purified collagenase type IV by A375 membrane fraction and membrane extract was also demonstrated by gelatin zymography. When A375 cells were first incubated with anti-MT1-MMP polyclonal antibody, activation of collagenase type IV was significantly decreased, indicating that membrane-associated MMP-2 activation is MT1-MMP-mediated. Immunocytochemistry showed MT1-MMP localization at focal adhesion sites. The presence of the components of activation complex-MT1-MMP and integrin alphavbeta3-were confirmed by Western blot, cell adhesion assay, and integrin subunit assay. CONCLUSION: Our experimental findings furnish another example of the unique membrane-associated MMP-2 activation mechanism in A375 melanoma cells and clearly indicate the role of MT1-MMP in MMP-2 activation. The information could help in developing new therapies designed to interfere with MMP activation and management of cancer and metastases.     

Polyhalide derivatives of poly[N-(2-aminoethyl)-acrylamido]triethylammonium resins as iodinating reagents

Poly[N-(2-aminoethyl)acrylamido]triethylammonium dichloroiodate, tetrachloroiodate and dibromoiodate resins have been developed as a new class of heterogeneous reagents. The use of the polyhalide derivatives for the selective iodination of aromatic amines and phenols is described. The influence of the various reaction parameters including nature of the solvent, effective molar concentration of the reagent and temperature were investigated to find out the optimum reaction conditions for achiveing selectivity for monoiodination.     

The potato virus X TGBp2 protein association with the endoplasmic reticulum plays a role in but is not sufficient for viral cell-to-cell movement

Potato virus X (PVX) TGBp1, TGBp2, TGBp3, and coat protein are required for virus cell-to-cell movement. Plasmids expressing GFP fused to TGBp2 were bombarded to leaf epidermal cells and GFP:TGBp2 moved cell to cell in Nicotiana benthamiana leaves but not in Nicotiana tabacum leaves. GFP:TGBp2 movement was observed in TGBp1-transgenic N. tabacum, indicating that TGBp2 requires TGBp1 to promote its movement in N. tabacum. In this study, GFP:TGBp2 was detected in a polygonal pattern that resembles the endoplasmic reticulum (ER) network. Amino acid sequence analysis revealed TGBp2 has two putative transmembrane domains. Two mutations separately introduced into the coding sequences encompassing the putative transmembrane domains within the GFP:TGBp2 plasmids and PVX genome, disrupted membrane binding of GFP:TGBp2, inhibited GFP:TGBp2 movement in N. benthamiana and TGBp1-expressing N. tabacum, and inhibited PVX movement. A third mutation, lying outside the transmembrane domains, had no effect on GFP:TGBp2 ER association or movement in N. benthamiana but inhibited GFP:TGBp2 movement in TGBp1-expressing N. tabacum and PVX movement in either Nicotiana species. Thus, ER association of TGBp2 may be required but not be sufficient for virus movement. TGBp2 likely provides an activity for PVX movement beyond ER association.     

Tremorogenesis by physostigmine is unrelated to acetylcholinesterase inhibition: evidence for serotoninergic involvement

Studies were performed to bring out a serotoninergic involvement in physostigmine tremor, hitherto known to be working via the cholinergic system. 5-Hydroxytryptamine (5-HT) was estimated fluorimetrically after isolation on Sephadex G-10 and acetylcholinesterase (AChE) was assayed spectrophotometrically. The dose-dependent tremor was quantified by a double-blind study. No correlation (r = 0.01) existed between tremor and AChE inhibition since the non-tremoring dose of physostigmine caused the same degree of enzyme inhibition. An increase of 5-HT was found to be correlated (r = 0.59) with the duration and intensity of tremor. Cholinergic antagonists atropine (2 and 5 mg/kg, i.p.), scopolamine (0.5, 1.0, 2.0 mg/kg, i.p.) and mecamylamine (1 mg/kg, i.p.) failed to block the tremor while the 5-HT antagonists methysergide (5 mg/kg, i.v.) and cyproheptadine (10 and 30 mg/kg, s.c.) could afford more than 60% protection. These results suggest a serotoninergic rather than a cholinergic component in the genesis of physostigmine tremor.     

Use of cuprous ion and metallic copper as initiators of aqueous vinyl polymerization

Cuprous chloride and copper powder (in acid media) initiate the aqueous polymerization of certain vinyl monomers such as methyl methacrylate, ethyl methacrylate, methacrylic acid and styrene but it fails in case of acrylonitrile, methyl acrylate, and acrylic acid under identical conditions. Catalyst exponent of this initiator (in acid aqueous media) with respect to methyl methacrylate is 0.38. Quantitative determination of end-groups by dye techniques of the polymers obtained shows that about 2 hydroxyl end-groups per chain are present under acidic conditions in presence of slight traces of oxygen but in rigorous absence of oxygen, very little OH end-groups are found incorporated. The mechanism of initiation under different experimental condition is discussed.