Tachykinin receptor cross-talk. Immunological cross-reactivity between the external domains of the substance K and substance P receptors.

In the present study, we have chemically synthesized peptides which correspond to the four putative extracellular domains of the predicted substance K (SK) receptor protein and raised specific polyclonal antibodies against these peptides. These antibodies were then tested for both structural and functional recognition of epitopes on the substance P (SP) receptor on rat AR42J pancreatic cells and human IM9 lymphoblasts, which express the SP receptor, but not the SK receptor. Antibodies directed against the first, second, and fourth external domains of the predicted SK receptor recognized a 58-kDa protein on AR42J cells. This protein has a molecular weight similar to the previously demonstrated SP receptor on both AR42J cells and IM9 cells. Furthermore, antibodies against the second and fourth extracellular domains significantly inhibited specific 125I-SP binding on both AR42J and IM9 cells, and also significantly inhibited SP-induced mobilization of [Ca2+]i on AR42J cells. These data suggest that the second and fourth extracellular domains of the SK and SP receptors may share common structural motifs for ligand binding and signaling mechanism.     

Solubilization and characterization of the pyrilamine-binding protein from cultured smooth muscle cells

The cultured smooth muscle cell line DDT1MF-2 expresses a large number (9.7 x 10(6) receptors/cell) of functional histamine H1-type receptors [J. Cell. Physiol. 134:367-375 1988]. Two different binding assays, gel filtration and polyethylene glycol precipitation, indicated that the [3H]pyrilamine binding activity was solubilized by 1% digitonin with binding characteristics similar to those of intact cells. The solubilized proteins were then purified by sequential gel filtration, chromatofocusing, and reverse phase high pressure liquid chromatography. The calculated molecular weight of this purified pyrilamine-binding protein was 38-40 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. [3H]Pyrilamine binding to these 38-40-kDa proteins indicated a single class of binding site with a Kd of 288 nM, which is equivalent to that of intact cells and digitonin-solubilized proteins. The computer analysis Scatfit also indicated that one molecule of [3H]pyrilamine bound to one molecule of purified protein. Furthermore, a polyclonal antibody raised against the purified protein recognized the 38-40-kDa band by Western blotting techniques, specifically bound to the cell surface of DDT1MF-2 cells, and inhibited [3H]pyrilamine binding to these cells in a dose-dependent manner. These data strongly suggest that the purified 38-40-kDa protein is part of an antagonist binding domain on the histamine H1 receptor on DDT1MF-2 cells.     

Intraocular pressure during vitrectomy in the diabetic patient

23 eyes underwent vitrectomy for diabetic proliferative retinopathy complications: vitreous hemorrhage with or without tractional retinal detachment. After 6 months of follow-up, 64% of eyes had final visual acuities of 1/40 or better. Preoperative iris neovascularization and preoperative detachment of the macula have a worse prognosis. Decrease of peroperative complications is allowed by checking of intraocular pressure during vitrectomy.     

Dynamic study of anti-HIV antibody avidity after cellular immunity restoration under antiretroviral treatment

We have studied the evolution of the avidity of anti-HIV antibodies, in 14 infected patients with Aids, including 11 patients with severe immunodeficiency at Aids stage and under active antiretroviral therapy (HAART), and 3 non-treated patients with moderate immunodeficiency. These patients have been followed up to 4 years, every 4 months the first year and every 6 months the three others, with HIV1 RNA viral load, CD4 and CD8 cells dosages and anti-HIV avidity measurements (Axsym HIV-1/2), using 1 M guanidine denaturation. A rapid decrease of the viral load was observed under Haart, inducing immune restoration with CD4 and CD8 cells increases (10 and 2-fold respectively). The decrease of anti-HIV avidity (- 20%) has been observed after 5 to 8 months under Haart, with a return to baseline value (84%). The quick restoration of CD4 cells with a persistence of viral antigens at the beginning of treatment has facilitated the selection of novel naive B lymphocytes producing low-affinity antibodies, measured by the decrease of global anti-HIV avidity. The reduction or even clearance of viral antigens under Haart could secondarily induce the selection of B lymphocytes with higher antibody affinity and therefore higher anti-HIV avidity. Thus, this avidity measurement could be used to assess the functional activity of CD4 cells restoration in HIV infected patients under Haart.     

Changes in immunoregulatory lymphocyte populations in patients with histoplasmosis

Circulating T-lymphocyte subpopulations were enumerated in 65 patients with histoplasmosis and correlated with the different clinical manifestations of the disease. Acute pulmonary histoplasmosis, rheumatologic, disseminated, and chronic inflammatory manifestations of histoplasmosis were all associated with a significant elevation above normal of OKT₈+ (suppressor-cytotoxic) lymphocytes and a significantly lower than normal OKT₄+ (helper-inducer)-lymphocyte to OKT₈+-lymphocyte ratio. In contrast, cavitary disease was associated with an increase in OKT₄+ lymphocytes, a decrease in OKT₈+ lymphocytes, and a higher than normal OKT₄/OKT₈ ratio. Clinical recovery was associated with normalization of these values. Functional activity determined by coculture techniques correlated closely with T-lymphocyte subset measurements. These distinct subset abnormalities may help monitor immunological aspects of disease activity.     

Antibody avidity: use for the diagnosis of HIV early infection

Determination of IgG avidity is useful to distinguish primary infection from reactivation or reinfection in viral, parasitic or bacterial infections. For diagnosis of HIV type 1 primary infection, the detection of IgM antibodies is often useless since they are also found in chronic infection. The usual serology (Elisa, western-blot, p24 antigen) may present no interest if done too late (more than 2 or 3 months after infection). Therefore, we have developed a test to determine the avidity of anti-HIV1 antibodies, using 1 M guanidine as denaturing agent. We have adapted the measurement of avidity to the Axsym automatic system for a routine use. Indeed, since requests for avidity determinations are sporadic, the use of microplates is not convenient. Using this assay, we found a low avidity (less than 50%) in immunocompetent and recent infected patients (less than 6 months), compared to old infected patients (more than 12 months) who had high avidity (80 to 100%). However, early treated patients (in the 6 months after contamination) had also low avidities but with a slower development of antibody maturation (8 to 27 months versus 2 to 8 months in non treated patients). To conclude, the determination of the anti-HIV1 avidity, according to the proper procedures explained here (notion of treatment and/or serious immunodepression), may help the physician to date the infection in each new infected patient who might benefit from an early treatment.     

Multiple mononeuropathy as the initial presentation of systemic lupus erythematosus — nerve biopsy and response to plasma exchange

Summary A total of 15 patients affected by idiopathic dystonia (7 with generalized and 8 with focal or segmental dystonia) were subjected to therapy with bromocriptine at low doses, pimozide and trihexyphenidyl. The symptoms were evaluated by giving a progressive score in relation to the intensity of the dystonic symptom to each of the body segments involved by the dystonia. Bromocriptine did not significantly modify the dystonia. Pimozide showed a slight nonsignificant improvement of the dystonic symptoms. Trihexyphenidyl was effective in the generalized dystonias, in agreement with previous reports in the literature. The variation in the pharmacological results could be due to the diversity of the dystonic syndromes, which comprise cases that are different in age at onset, site of dystonic symptoms, and evolution.

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Zusammenfassung Fünfzehn von idiopathischer — und zwar 7 von generalisierter und 8 von fokaler und segmentarischer — Dystonie befallenen Patienten unterzogen sich verschiedenen pharmakologischen Behandlungen mit kleinen Mengen Bromocriptine, Pymozide und Triesifenidile. Die Symptome wurden durch eine fortlaufende Punktzahl bezeichnet, so daß deren Schätzung von der Intensität des Symptoms Dystonie in jedem einzelnen befallenen Körperteil abhing. Die Dystonien wurden durch Bromocriptine nicht bedeutend geändert.Pymozide führte zu einer geringeren, doch unbedeutenden, Besserung der dystonischen Symptome.Triesifenidile wirkte auf die generalisierten Dystonien, in Übereinstimmung mit einigen Literaturangaben.Die Veränderlichkeit der pharmakologischen Ergebnisse wurde auf die Verschiedenheit der dystonischen Syndrome zurückgeführt, unter denen man Fälle versammelt, die sich durch Anfangsalter, Sitz der dystonischen Symptome und Entwicklungsart voneinander unterscheiden.

     

Utility of peptide–protein affinity complexes in proteomics: identification of interaction partners of a tumor suppressor peptide*

Abstract: We used a N‐biotinylated peptide analog of the C‐terminal domain of the tumor suppressor protein, p21^cip1/waf1 to elucidate peptide/protein interacting partners. The C‐terminal domain of p21^cip1/waf1 protein spanning 141–160 amino acid residues is known to bind PCNA and this interaction is important in many biological processes including cell‐cycle control. This C‐terminal 20‐mer efficiently extracts PCNA in the presence of a variety of N‐ or C‐terminally attached affinity tags. Using difference silver stained 2D gels combined with in‐gel tryptic digests, we identified the difference spots using MALDI‐TOF mass spectrometry‐based peptide mass fingerprinting followed by a database search using profound against NCBIs human nonredundant protein sequence data bank. Identified spots include the p48 subunit of chromatin assembly factor‐1, the heat shock 70 protein analog BiP, calmodulin, nucleolin and a spot similar in size to dimeric PCNA. In contrast, microcapillary ion‐trap LC‐MS/MS analysis of a tryptic digest of entire affinity extracts derived from both control and experimental runs followed by database searches using sequest confirmed the presence of most of the above proteins. This strategy also identified hnRNPA1, HPSP90α, HSP40 and T‐complex protein 1, a protein similar to prothymosin, and a possible allelic variant of the p21^cip1/waf1 protein. The use of N‐biotinylated peptide derived from the C‐terminal domain of p21^cip1/waf1 protein in proteomic analysis exemplified here suggests that peptides obtained from intracellular functional screens could also potentially serve as efficient baits to discover new drug targets.