Cooperative & competitive binding of anti-myosin tail antibodies revealed by super-resolution microscopy.

The MF30 monoclonal antibody, which binds to the myosin subfragment-2 (S2), was found to increase the extent of myofibril shortening. Yet, previous observations found no effect of this antibody on actin sliding over myosin during in vitro motility assays with purified proteins in which myosin binding protein C (MyBPC) was absent. MF30 is hypothesized to enhance the availability of myosin heads (subfragment-1 or S1) to bind actin by destabilizing the myosin S2 coiled-coil and sterically blocking S2 from binding S1. The mechanism of action likely includes MF30's substantial size, thereby inhibiting S1 heads and MyBPC from binding S2. Hypothetically, MF30 should enhance the ON state of myosin, thereby increasing muscle contraction. Our findings indicate that MF30 binds preferentially to the unfolded heavy chains of S2, displaying positive cooperativity. However, the dose-response curve of MF30's enhancement of myofibril shortening did not suggest complex interactions with S2. Single, double, and triple-stained myofibrils with increasing amounts of antibodies against myosin rods indicate a possible competition with MyBPC. Additional assays revealed decreased fluorescence intensity at the C-zone (central zone in the sarcomere, where MyBPC is located), where MyBPC may inhibit MF30 binding. Another monoclonal antibody named MF20, which binds to the light meromyosin (LMM) without affecting myofibril contraction, showed less reduction in fluorescence intensity at the C-zone in expansion microscopy than MF30. Expansion microscopy images of myofibrils labeled with MF20 revealed labeling of the A-band (anisotropic band) and a slight reduction in the labeling at the C-zone. The staining pattern obtained from the expansion microscopy image was consistent with images from photolocalization microscopy which required the synthesis of unique photoactivatable quantum dots, and Zeiss Airyscan imaging as well as alternative expansion microscopy digestion methods. Consistent with the hypothesis that MF30 competes with MyBPC binding to S2, cardiac tissue from MyBPC knockout mice was stained more intensely, especially in the C-zone, by MF30 compared to the wild type.

The antibiotics doxycycline and minocycline inhibit the inflammatory responses to the Lyme disease spirochete Borrelia burgdorferi.

Tetracyclines moderate inflammatory responses of various etiologies. We hypothesized that tetracyclines, in addition to their antimicrobial function, could exert control over the inflammation elicited by Borrelia burgdorferi. To model systemic effects, we used the human monocytic cell line THP-1; to model effects in the central nervous system, we used rhesus monkey brain astrocytes and microglia. Cells were stimulated with live or sonicated B. burgdorferi or with the lipoprotein outer surface protein A in the presence of increasing concentrations of doxycycline or minocycline. Both antibiotics significantly reduced the production of tumor necrosis factor-alpha, interleukin (IL)-6, and IL-8 in a dose-dependent manner in all cell types. Microarray analyses of the effect of doxycycline on gene transcription in spirochete-stimulated monocytes revealed that the NFKB and CHUK (alias, IKKA) genes were down-regulated. Functionally, phosphorylation of IkappaBalpha and binding of NF-kappaB to target DNA were both reduced in these cells. Our results suggest that tetracyclines may have a dual therapeutic effect in Lyme disease.

Toll-like receptors: insights into their possible role in the pathogenesis of lyme neuroborreliosis.

Lyme neuroborreliosis is likely caused by inflammatory effects of the tick-borne spirochete Borrelia burgdorferi on the nervous system. Microglia, the resident macrophage cells within the central nervous system (CNS), are important in initiating an immune response to microbial products. In addition, astrocytes, the major CNS glial cell type, also can contribute to brain inflammation. TLRs (Toll-like receptors) are used by glial cells to recognize pathogen-associated molecular patterns (PAMPs), mediate innate responses, and initiate an acquired immune response. Here we hypothesize that because of their PAMP specificities, TLR1, -2, -5, and -9 may be involved in the pathogenesis of Lyme neuroborreliosis. Previous reports have shown that the rhesus monkey is the only animal model to exhibit signs of Lyme neuroborreliosis. Therefore, we used primary cultures of rhesus astrocytes and microglia to determine the role of TLRs in mediating proinflammatory responses to B. burgdorferi. The results indicate that microglia and astrocytes respond to B. burgdorferi through TLR1/2 and TLR5. In addition, we observed that phagocytosis of B. burgdorferi by microglia enhances not only the expression of TLR1, -2, and -5, but also that of TLR4. Taken together, our data provide proof of the concept that astrocyte and microglial TLR1, -2, and -5 are involved in the in vivo response of primate glial cells to B. burgdorferi. The proinflammatory molecules elicited by these TLR-mediated responses could be a significant factor in the pathogenesis of Lyme neuroborreliosis.

Characterization of receptors for growth hormone-releasing hormone in human osteosarcomas and Ewing's sarcomas.

Antagonists of growth hormone-releasing hormone (GH-RH) inhibit growth of various human cancers including osteosarcomas and Ewing's sarcomas, xenografted into nude mice or cultured in vitro. The antiproliferative effect of GH-RH antagonists could be mediated, in part, through the splice variants (SVs) of receptors for GH-RH which have been found in several human cancers and cancer cell lines. In this study we investigated the expression of SVs of GH-RH receptors and the binding characteristics of these receptor isoforms in MNNG/HOS human osteosarcoma and SK-ES-1 human Ewing's sarcoma grown in nude mice. RT-PCR revealed the presence of mRNA for SVs of GH-RH receptors in both human malignant bone cancer models. Using ligand competition assays with 125I-labeled GH-RH antagonist JV-1-42, we demonstrated in MNNG/HOS and SK-ES-1 tumors the presence of specific high affinity binding sites for GH-RH (Kd=5.83 nM and Kd=2.76 nM) with a maximal binding capacity (Bmax) of 552.1 fmol/mg protein and 371.9 fmol/mg protein, respectively. We also investigated the effect of GH-RH antagonist JV-1-38, administered s.c. at a dose of 20 microg twice daily for 4 weeks on the gene expression, affinity and concentration of receptors for GH-RH in MNNG/HOS human osteosarcomas xenografted into nude mice. Treatment with JV-1-38 did not affect the expression and binding characteristics of GH-RH receptors. High affinity binding of JV-1-38 to GH-RH receptors on MNNG/HOS tumors was characterized by an IC50 value of 1.04 nM. The presence of GH-RH receptors in human bone tumors provides a rationale for new approaches to the therapy of this malignancy based on GH-RH antagonists.

Inhibition of mutant p53 expression and growth of DMS-153 small cell lung carcinoma by antagonists of growth hormone-releasing hormone and bombesin.

We investigated the effects of growth hormone-releasing hormone (GHRH) antagonists, JV-1-65 and JV-1-63, and bombesin/gastrin-releasing peptide (BN/GRP) antagonist RC-3940-II on DMS-153 human small cell lung carcinoma xenografted into nude mice. Treatment with 10 microg/day JV-1-65 or RC-3940-II decreased tumor volume by 28% (P < 0.05) and 77% (P < 0.01), respectively, after 42 days compared with controls. Combination of JV-1-65 and RC-3940-II induced the greatest inhibition of tumor proliferation (95%; P < 0.01), suggesting a synergism. Western blotting showed that the antitumor effects of these antagonists were associated with inhibition of the expression of the mutant tumor suppressor protein p53 (Tp53). Mutation was detected by sequence analysis of the p53 gene at codon 155: ACC [Thr] --> CCC [Pro]. Combination of JV-1-65 and RC-3940-II decreased the levels of mutant p53 protein by 42% (P < 0.01) compared with controls. JV-1-65, JV-1-63, and RC-3940-II, given singly, reduced mutant p53 protein expression by 18-24% (P < 0.05). Serum insulin-like growth factor (IGF)-I levels were diminished in animals receiving GHRH antagonists. mRNA levels for IGF-II, IGF receptor-I, GRP receptor, and EGF receptor in tumors were significantly decreased by combined treatment with JV-1-65 and RC-3940-II. DMS-153 tumors expressed mRNAs for GHRH and GHRH receptor splice variants 1 and 2, suggesting that GHRH could be an autocrine growth factor. Proliferation of DMS-153 cells in vitro was stimulated by GRP and IGF-II and inhibited by JV-1-65. This study indicates that GHRH antagonists and BN/GRP antagonist inhibit the growth of DMS-153 small cell lung carcinoma concomitantly with the expression of mutant Tp53, which might uncouple the signal transduction pathways for cell growth stimulation.

CFTR, PRSS1 and SPINK1 mutations in the development of pancreatitis in Brazilian patients.

CONTEXT: Mutations in cystic fibrosis transmembrane conductance regulator (CFTR), in cationic trypsinogen (PRSS1) and in serine protease inhibitor Kazal type 1 (SPINK1) genes have been associated with chronic pancreatitis (alcohol related, idiopathic and hereditary). However, the inheritance pattern is still not clear. PATIENTS: Eighty-two unrelated Brazilian patients with chronic pancreatitis (alcohol-related disease in 64, idiopathic disease in 16, and hereditary disease in 2). Two hundred unrelated individuals with an ethnic distribution comparable to the patients were studied as controls. MAIN OUTCOME MEASURE: Detection of mutations in CFTR, PRSS1, and SPINK1 genes. RESULTS: Mutations in the CFTR gene were found in 8 patients (9.8%) with chronic pancreatitis, 5 of them with idiopathic disease. Interestingly, the only clinical symptom in a male patient in the alcoholic group, who was a compound heterozygote (DeltaF508/R170C) for two CFTR mutations, was pancreatitis without infertility or pulmonary involvement. In the PRSS1 gene, the E79K change in exon 3 was found in one patient (1.2%) with alcohol-related chronic pancreatitis. Four different alterations were identified in the SPINK1 gene. CONCLUSIONS: Mutations in the CFTR gene represent the major cause of idiopathic chronic pancreatitis in Brazilian patients. No mutation was found in the PRSS1 gene among our patients suggesting further genetic heterogeneity for hereditary and idiopathic chronic pancreatitis. Interestingly, the most frequent SPINK1 N34S mutation was not present in patients or controls. Moreover, the -253C allele for the SPINK1 gene was significantly more frequent in patients than controls (P=0.004), suggesting that it might represent a risk factor for the development of pancreatitis in our population.

Analysis of mutations in the cystic fibrosis transmembrane regulator (CFTR) gene in patients with obstructive azoospermia

Congenital bilateral absence of the vas deferens (CBAVD) accounts for 1 per cent-2 per cent of sterility in men. A high incidence of mutations, as well as the involvement of the 5T variant of the T tract length in intron 8 of the cystic fibrosis conductance regulator (CFTR) gene, have been previously described in males with CBAVD. Herein we report the screening for mutations and for the 5T variant of the CFTR gene in 17 patients with CBAVD and three others with non-CABVD obstructive azoospermia. In the CBAVD group, three patients (15 per cent) were compound heterozygotes for mutations, and five patients (25 per cent) had a mutation in one allele and the 5T variant in the other; the 5T variant was also present in two other patients, one of them being homozygous. The most frequent mutation was DeltaF508, present on five chromosomes (12.5 per cent). A novel missense mutation (A399D) was detected in a Japanese CBVAD patient. Our results yield further evidence for a strong association between male obstructive azoospermia caused by CBAVD and mutation/5T variant in the CFTR gene. The search for CFTR mutations in such patients is thus recommended for genetic counseling of couples who undergo assisted fertilization due to CBAVD