Human amniotic fluid-derived c-kit(+) and c-kit (-) stem cells: growth characteristics and some differentiation potential capacities comparison

Amniotic fluid (AF) contains heterogeneous and multipotential cell types. A pure mesenchymal stem cells group can be sorted from AF using flow cytometry. In order to evaluate a possible therapeutic application of these cells, the human AF-derived c-kit⁺ stem cells (c-kit⁺ AFS) were compared with the c-kit⁻ (unselected) stem cells (c-kit⁻ AFS). Our findings revealed that the optimal period to obtain c-kit⁺ AFS cells was between 16 and 22 weeks of gestation. Following cell sorting, c-kit⁺ AFS cells shared similar morphological and proliferative characteristics as the c-kit⁻ AFS cells. Both c-kit⁺ and c-kit⁻ AFS cells had the characteristics of mesenchymal stem cells through surface marker identification by flow cytometric and immunocytochemical analysis. Both c-kit⁺ and c-kit⁻ AFS cells could differentiate along adipogenic and osteogenic lineages. However, the myocardial differentiation capacity was enhanced in c-kit⁺ AFS cells by detecting GATA-4, cTnT, α-actin, Cx43 mRNA and protein expression after myocardial induction; whereas induced c-kit⁻ AFS cells were only detected with GATA-4 mRNA and protein expression. The c-kit⁺ AFS cells could have potential clinical application for myogenesis in cardiac regenerative therapy.     

Antigen-binding activity,structural characteristics and use of monoclonal antibodies to human alpha-1-microglobulin

Four monoclonal antibodies (mAbs), G6, F9, H8, and B2, against human alpha-1-microglobulin (A1M) have been produced and characterized. The parameters of affinity (K_p ～ 10⁹ M^?1), epitope specificity (the additively binding G6/F9, G6/H8, G6/B2, F9/H8, and F9/B2 pairs), and the observed effect of reversibility of structural changes induced by chemical agents allow use of these mAbs in biospecific methods of A1M purification and quantitative determination. The application of mAbs to an A1M enzyme immunoassay (analytical sensitivity—0.5 μg/l) and one step isolation of pure A1M by immunoaffinity chromatography was described.     

Biochemical and antigenic analysis of the Epstein Barr virus/C3d receptor (CR2)

Four monoclonal antibodies (OKB7, HB-5, AB-1, and anti-B2) that recognize a 145-kDa B cell-specific membrane structure have markedly different abilities to 1) inhibit C3d and EBV binding to B cells, 2) immunoprecipitate a 145-kDa B cell protein, and 3) stimulate B cell proliferation and differentiation into Ig-secreting cells. This study was initiated to determine whether these four monoclonal antibodies (MoAb) react with the same protein; a related goal was to determine whether the structure(s) recognized by these antibodies constitutes an antigenically related family of structurally distinct molecules. In the studies presented here, the four MoAb were found to fully immunoprecipitate the purified 145-kDa B cell molecule isolated by immunoaffinity chromatography on either OKB7, HB-5, or AB-1 columns, findings that show conclusively that the antibodies all react with the same B cell protein. The variable ability to immunoprecipitate this B cell membrane protein was found to result from differences in exposure or accessibility of the relevant antigenic epitopes in the detergent extract. The 145-kDa molecule immunoprecipitated with the four MoAb was equivalently sensitive to endoglycosidase F and yielded the same banding pattern after digestion with endoglycosidase F and after partial digestion with either S. aureus V8 protease or with trypsin. Within the limits of the sensitivity of these techniques, therefore, there is no evidence for carbohydrate or protein differences in the EBV/C3d receptor (CR2) molecule recognized by the four MoAb. Additional studies showed that the four MoAb react with distinct and nonoverlapping antigenic epitopes on the 145-kDa molecule. The variable abilities of the four MoAb to inhibit CR2 function and EBV binding and to trigger B cell activation, together with the other findings noted above, indicates that the 145-kDa EBV/C3d receptor possesses discretely localized functional domains.     

Allosteric effects of monoclonal antibodies on human growth hormone

We have previously shown that a monoclonal antibody (MAb) recognizing the human growth hormone (hGH) antigenic domain left exposed after binding to lactogenic receptors enhanced hGH binding probably through allosteric effects on the hormone binding site. Since receptors displaying different specificities would not recognize exactly the same hGH region, we explored whether some of our MAb could affect hGH binding to somatogenic receptors from rabbit liver and to human liver hGH-specific receptors.The effect of MAbAE5, AC8 and F11 on hGH binding was measured by determining the formation of¹²⁵I-MAb:hGH:receptor complexes using two different experimental approaches. Results from procedure A, which involved the previous binding of the hormone to microsomes before adding¹²⁵I-MAb, indicated that the hGH domain defined by epitopes AE5, AC8 and F11 is uncovered in the various hormone:receptor complexes.Procedure B was devised to reveal any alteration in the hGH molecule induced by the MAb. In this case preformed¹²⁵I-MAb:hGH complexes were added to microsomes. Data showed that¹²⁵I-MAb AE5:hGH complexes bound better to the various receptors than¹²⁵I-MAb AE5 to hGH:receptor complexes. On the contrary, hGH previously bound to¹²⁵I-MAb AC8 or¹²⁵I-MAb F11 was less recognized by the receptors than the free hormone. Furthermore, binding of MAb AE5 or MAb F11 to hGH 20 K (a natural hGH variant lacking residues 32–46) also enhanced its affinity to the various receptors whereas MAb AC8 did not inhibit hGH 20 K binding.Results indicated that MAb recognizing the hGH antigenic area that remains unmasked after binding to different membrane-bound receptors are able to affect hormone binding site. MAb would induce either positive or negative allosteric changes in the hormone region involved in its binding to lactogenic, somatogenic and hGH-specific receptors.     

Investigations into the synthesis,radiofluorination and conjugation of a new [18F]fluorocyclobutyl prosthetic group and its in vitro stability using a tyrosine model system

The [¹⁸F]fluorocyclobutyl group has the potential to be a metabolically stable prosthetic group for PET tracers. The synthesis of the radiolabeling precursor cis-cyclobutane-1,3-diyl bis(toluene-4-sulfonate) 8 was obtained from epibromohydrin in 7 steps (2% overall yield). The radiolabeling of this precursor 8 and its conjugation to l-tyrosine as a model system was successfully achieved to give the new non-natural amino acid 3-[¹⁸F]fluorocyclobutyl-l-tyrosine (L-3-[¹⁸F]FCBT) [¹⁸F]17 in 8% decay-corrected yield from the non-carrier-added [¹⁸F]fluoride. L-3-[¹⁸F]FCBT was investigated in vitro in different cancer cell lines to determine the uptake and stability. The tracer [¹⁸F]17 showed a time dependent uptake into different tumor cell lines (A549, NCI-H460, DU145) with the best uptake of 5.8% injected dose per 5 × 10⁵ cells after 30 min in human lung carcinoma cells A549. The stability of L-3-[¹⁸F]FCBT in human and rat plasma and the stability of the non-radioactive L-3-FCBT in rat hepatocytes were both found to be excellent. These results show that the non-natural amino acid L-3-[¹⁸F]FCBT is a promising metabolically stable radiotracer for positron emission tomography.     

Human immunodeficiency virus type 1 gp41 antibodies that mask membrane proximal region epitopes: antibody binding kinetics, induction, and potential for regulation in acute infection

Two human monoclonal antibodies (MAbs) (2F5 and 4E10) against the human immunodeficiency virus type 1 (HIV-1) envelope g41 cluster II membrane proximal external region (MPER) broadly neutralize HIV-1 primary isolates. However, these antibody specificities are rare, are not induced by Env immunization or HIV-1 infection, and are polyspecific and also react with lipids such as cardiolipin or phosphatidylserine. To probe MPER anti-gp41 antibodies that are produced in HIV-1 infection, we have made two novel murine MAbs, 5A9 and 13H11, against HIV-1 gp41 envelope that partially cross-blocked 2F5 MAb binding to Env but did not neutralize HIV-1 primary isolates or bind host lipids. Competitive inhibition assays using labeled 13H11 MAb and HIV-1-positive patient plasma samples demonstrated that cluster II 13H11-blocking plasma antibodies were made in 83% of chronically HIV-1 infected patients and were acquired between 5 to 10 weeks after acute HIV-1 infection. Both the mouse 13H11 MAb and the three prototypic cluster II human MAbs (98-6, 126-6, and 167-D) blocked 2F5 binding to gp41 epitopes to variable degrees; the combination of 98-6 and 13H11 completely blocked 2F5 binding. These data provide support for the hypothesis that in some patients, B cells make nonneutralizing cluster II antibodies that may mask or otherwise down-modulate B-cell responses to immunogenic regions of gp41 that could be recognized by B cells capable of producing antibodies like 2F5.     

YB‐1 orchestrates onset and resolution of renal inflammation via IL10 gene regulation

The Y‐box‐binding protein (YB)‐1 plays a non‐redundant role in both systemic and local inflammatory response. We analysed YB‐1‐mediated expression of the immune regulatory cytokine IL‐10 in both LPS and sterile inflammation induced by unilateral renal ischaemia–reperfusion (I/R) and found an important role of YB‐1 not only in the onset but also in the resolution of inflammation in kidneys. Within a decisive cis‐regulatory region of the IL10 gene locus, the fourth intron, we identified and characterized an operative YB‐1 binding site via gel shift experiments and reporter assays in immune and different renal cells. In vivo, YB‐1 phosphorylated at serine 102 localized to the fourth intron, which was paralleled by enhanced IL‐10 mRNA expression in mice following LPS challenge and in I/R. Mice with half‐maximal expression of YB‐1 (Yb1^+/?) had diminished IL‐10 expression upon LPS challenge. In I/R, Yb1^+/? mice exhibited ameliorated kidney injury/inflammation in the early‐phase (days 1 and 5), however showed aggravated long‐term damage (day 21) with increased expression of IL‐10 and other known mediators of renal injury and inflammation. In conclusion, these data support the notion that there are context‐specific decisions concerning YB‐1 function and that a fine‐tuning of YB‐1, for example, via a post‐translational modification regulates its activity and/or localization that is crucial for systemic processes such as inflammation.     

Three Antigenic Regions in p17 of Human Immunodeficiency Virus Type 1 (HIV-1) Revealed by Mouse Monoclonal Antibodies and Human Antibodies in HIV-1 Carrier Sera

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (K_A = 1.9 × 10⁵?1.4 × 10⁸ M^?1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (K_A = 2.0 × 10⁵?1.8 × 10⁷ M^?1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.     

Monoclonal antibodies to alpha-endorphin effective in immunohistochemistry and immunoblotting

Four mouse monoclonal antibodies (E11, A8, F8, H5) to alpha-endorphin have been produced. The antibodies bind 12.5, 20.6, 9.6, 6.6% of 125I-beta-endorphin and 35.5, 15.1, 12.8, 12.2% of 125I-gamma-endorphin; the binding of 125I-alpha-endorphin being taken for 100%. The binding of antibodies E11, A8, F8 and H5 to 125I-alpha-endorphin was 50% inhibited by unlabeled ligand in concentrations 5, 50, 30 and 35 nM respectively. Using tissue sections of rat pituitary it was shown that antibody E11 can be used for the localization of endorphin producing cells by immunofluorescence. The antibodies F8 and H5 effectively detected endorphin precursor proopiomelanocortin by immunoblotting.     

The detection of virally induced tumors by 131I- and 125I-labeled syngeneic monoclonal antibodies

Summary The binding of the syngeneic monoclonal antibodies IC5F5 and 4D2B4 to Rauscher virus-induced myeloid leukemic (RMB-1) cells was analyzed in vivo in tumor-bearing BALB/c mice. To verify it these antibodies bind specifically to RMB-1 cells, purified antibodies were iodinated with the isotopes ¹²⁵I and ¹³¹I. Mice previously inoculated with tumor cells were injected with these labeled monoclonal antibodies and the plasma clearance and the tissue distribution were determined. The clearance in tumor-bearing animals was faster than in control mice. The tissue distribution was corrected for nonspecific accumulation by scoring for an unrelated antibody. Calculation of a localization index showed that IC5F5 binds at least 4.5 times more specifically to tumor cells than to other tissues. A preferential localization of radioactivity in s.c. tumor tissue was seen in the scanning of animals injected with ¹³¹I-labeled antibodies. The most direct proof of specific binding was observed in autoradiograms of animals treated with ¹²⁵I-labeled antibodies. Small islands of tumor cells in the livers of mice inoculated i.v. had a high density of grains compared to other tissues and also compared to tumor cells in mice treated with unrelated monoclonal antibodies. These results show efficient targeting of these monoclonal antibodies and make immunotherapy of these myeloid leukemic cells possible.     

Expression of CD15 (FAL) on myeloid cells and chromosomal localization of the gene

Summary Different CD15 murine monoclonal antibodies were studied. These antibodies appeared to react specifically with the human myeloid-lineage-derived cell types in both peripheral blood and bone marrow.The antigens recognized by these antibodies were immunoprecipitated from lysates of¹²⁵I-labelled neutrophilic PMNs of healthy donors and subsequently analysed by electrophoresis on SDS-polyacrylamide gel and autoradiography. All antibodies precipitated the same membrane polypeptides from the membrane-iodinated PMN lysates: 105 and 150-kDa as most prominent, together with 260-, 230-, 67- and 52-kDa polypeptides.Absorption studies were performed with synthesized carbohydrate molecules. Antibody B4.3 appears to be directed against 3--fucosyl-N-acetyl-lactosamine (FAL). Competition experiments with¹²⁵I-labelled B4.3 demonstrated complete inhibition of binding by B4.3 and three other CD15 antibodies (VIM D5, UJ308, MI/N1), and partial inhibition by three additional antibodies (FMC10, FMC12, FMC13), indicating binding to the same antigenic structure. None of the antibodies reacted with monocytes using the immunofluorescence technique, but after neuraminidase digestion of these cells, positive reactions were obtained with all antibodies. Immunoprecipitation with lysates of both native and neuraminidase-digested monocytes showed no polypeptide bands. Monocytic differentiation of the myeloid cell line HL60 by 12-O-tetradecanoylphorbol-13-acetate (TPA) was accompanied by a decrease in reactivity with the antibodies, which could be reversed by neuraminidase digestion. This indicates that 3--fucosyl-N-acetyl-lactosamine is masked for the detection with antibodies upon monocytic differentiation by sialylation.Human x mouse myeloid cell hybrids were obtained after fusion of human myeloid cells and the HPRT-deficient murine myeloid cell line WEHI-TG. These hybrids were tested for reactivity with the anti-CD15 antibodies.The CD15 panel exhibited very similar reactivity patterns with the hybrid clones. Chromosomal analysis of hybrid cell metaphases revealed that the gene(s) involved in the expression of FAL must be located on human chromosome 11 in the region q12-qter.     

Pollen S–locus F–box proteins of Petunia involved in S–RNase‐based self‐incompatibility are themselves subject to ubiquitin‐mediated degradation

Many flowering plants show self‐incompatibility, an intra‐specific reproductive barrier by which pistils reject self‐pollen to prevent inbreeding and accept non‐self pollen to promote out‐crossing. In Petunia, the polymorphic S–locus determines self/non‐self recognition. The locus contains a gene encoding an S–RNase, which controls pistil specificity, and multiple S‐locus F‐box (SLF) genes that collectively control pollen specificity. Each SLF is a component of an SCF (Skp1/Cullin/F‐box) complex that is responsible for mediating degradation of non‐self S‐RNase(s), with which the SLF interacts, via the ubiquitin–26S proteasome pathway. A complete set of SLFs is required to detoxify all non‐self S‐RNases to allow cross‐compatible pollination. Here, we show that SLF1 of Petunia inflata is itself subject to degradation via the ubiquitin–26S proteasome pathway, and identify an 18 amino acid sequence in the C‐terminal region of S₂‐SLF1 (SLF1 of S₂ haplotype) that contains a degradation motif. Seven of the 18 amino acids are conserved among all 17 SLF proteins of S₂ haplotype and S₃ haplotype involved in pollen specificity, suggesting that all SLF proteins are probably subject to similar degradation. Deleting the 18 amino acid sequence from S₂‐SLF1 stabilized the protein but abolished its function in self‐incompatibility, suggesting that dynamic cycling of SLF proteins is an integral part of their function in self‐incompatibility.     

Isolation of a gene essential for biosynthesis of the lipopeptide antibiotics plipastatin B1 and surfactin in Bacillus subtilis YB8

Bacillus subtilis YB8 was found to produce the lipopeptide antibiotics surfactin and plipastatin B1. A gene, lpa-8, required for the production of both lipopeptides was cloned from strain YB8. When this gene was inactivated in strain YB8, neither surfactin nor plipastatin B1 was produced. However, the defective strain transformed with an intact lpa-8 gene had restored ability to produce both peptides. Nucleotide sequence analysis of the region essential for the production of the peptides revealed the presence of a large open reading frame. The deduced amino acid sequence of lpa-8 (224 amino acid residues) showed sequence similarity to that of sfp (from surfactin-producing B. subtilis), lpa-14 (from iturin A- and surfactin-producing B. subtilis), psf-1 (from surfactin-producing Bacillus pumilus), gsp (from gramicidin-S-producing Bacillus brevis), and entD (from siderophore-enterobactin-producing Escherichia coli), which are able to complement a defect in the sfp gene and promote production of the lipopeptide antibiotic surfactin. The sequence similarity among these proteins and the product similarity of cyclic peptides suggests that they might be involved in the biosynthesis or secretion of the peptides. Received: 14 July 1995 / Accepted: 22 December 1995     

A Salmonella typhimurium mutant unable to utilize fatty acids and citrate is avirulent and immunogenic in mice

Salmonella typhimurium SR-11 is extremely virulent at a dose as low as 10⁵ colony forming units (cfu) when administered perorally to BALB/c mice. Utilizing mini-transposon mutagenesis, a mutant of S. typhimurium SR-11 was isolated that was unable to utilize oleate and citrate as carbon sources. This mutant, designated S. typhimurium SR-11 Fad⁻ (Fatty acid), was found to utilize sugars under cya/crp control as sole carbon sources, suggesting that the mutation is not in either of these genes. In addition, SR-11 Fad⁻ utilized pyruvate and succinate, but was unable to utilize either acetate or isocitrate as sole carbon source. In contrast to SR-11, SR-11 Fad⁻ was found to be avirulent, i.e. BALB/c mice were completely healthy after oral infection with 10⁹ S. typhimurium SR-11 Fad⁻ cells. Moreover, 21 days after SR-11 Fad⁻ infection, BALB/c mice were found to be protected against an oral challenge with 10⁹ cells of S. typhimurium SR-11.     

Factors affecting pre-ovulatory follicle diameter in the mare: the effect of mare age, season and presence of other ovulatory follicles (multiple ovulation)

The importance of elucidating factors affecting reproductive performance and efficiency is of paramount concern to the equine industry. Oocyte viability is known to be one of the determinants of reproductive success and evidence suggests that it may be linked to follicle size. The aims of this study were, therefore, to ascertain: i) the average diameter and range of pre-ovulatory follicles in Thoroughbred mares; ii) whether this is affected by either mare age, time within the breeding season, or the presence of multiple pre-ovulatory follicles (MO). One thousand, four hundred and ninety two Thoroughbred mares, aged 2-26 years, were examined with ultrasound to ascertain ovulation date to within 24h, and pre-ovulatory follicle(s) (F1) diameter. Mares were divided into groups according to age (7 groups, 2-4 yr, 5-7 yr, 8-10 yr, 11-13 yr, 14-16 yr, 17-19 yr, >19 yr), time within the season (16 half-month groups, from Feb 1^st to Sept 30^th), and pre-ovulatory follicles (single, {SO} or multiple {MO}). Overall average F1 diameter was 39.95 ± 4.84 mm (range 22-50 mm). Mare age had a significant (P < 0.001) negative effect on F1 diameter (largest F1 38.95 ± 5.61 mm, mares 2-4 yrs; smallest F1 33.30 ± 4.66 mm, mares >19 yrs) as did season (largest F1 44.20 ± 3.95 mm, Feb 1^st-14^th; smallest F1 33.74 ± 4.87 mm, Aug 15^th-31^st) and the presence of more than one pre-ovulatory follicle (MO F1 35.45 ± 4.53 mm; SO F1 37.44 ± 4.84 mm). In conclusion older mares, bred towards the end of the breeding season, especially if MO were present, were more likely to ovulate from smaller follicles. If, as suggested, small pre-ovulatory follicle size is associated with low oocyte viability, then this may account, at least in part, for the poor fertility rates characteristic of older MO mares, bred later in the season and so justify increased monitoring and careful reproductive management of such mares.     

Dexamethasone downregulates expression of several genes encoding orphan nuclear receptors that are important to steroidogenesis in stallion testes

Glucocorticoids impair testosterone synthesis by an unknown mechanism. Stallions treated with the synthetic glucocorticoid dexamethasone had testes collected at 6 or 12 hours postinjection. The testicular expression of selected genes encoding nuclear receptors and steroidogenic enzymes was measured. At 6 hours, dexamethasone treatment decreased levels of NR0B2, NR4A1, NR5A1, and NR5A2 messenger RNAs (mRNAs) and NR5A2 mRNA levels remained depressed at 12 hours. In contrast, dexamethasone increased levels of NFKBIA mRNA at both time points. At 6 hours, dexamethasone did not alter levels of NR0B1, NR2F1, NR2F2, NR3C1, CYP11A1, CYP17A1, CYP19A1, DHCR24, GSTA3, HSD3B2, HSD17B3, LHCGR, or STAR mRNAs. In primary cultures of Leydig cells, 10 ^?9 and 10 ^?7M dexamethasone decreased levels of NR4A1 and NR5A1 mRNAs and increased those of NFKBIA mRNA. Our discovery that dexamethasone downregulates NR4A1, NR5A1, and NR5A2 genes, known to be important for testicular functions, may be part of the mechanism by which glucocorticoids acutely decreases testosterone.     

Degradation of nicosulfuron by Bacillus subtilis YB1 and Aspergillus niger YF1

The optimal degrading conditions for the nicosulfuron degradation by Bacillus subtilis YB1 and Aspergillus niger YF1, and site of their action on nicosulfuron were studied. The results showed that the degradation efficiency of free cells of B. subtilis YB1 and A. niger YF1 was respectively 87.9 and 98.8% in basic medium III containing 2 mg/l of nicosulfuron after inoculation with 1 ml of culture containing 2.3 × 10⁷ CFU ml^?1 and incubation for 5 days at 35°C. Moreover, the degradation rate of nicosulfuron by the mixture of microorganisms was much higher than for every of them taken separately in the same conditions. The mass spectrometric analysis of the products degraded by B. subtilis YB1 revealed that the sulfonylurea bridge in nicosulfuron molecule had been broken. Extracellular (EXF) and endocellular (ENF) fractions obtained from bacterium and fungus were tested for the ability to degrade nicosulfuron. The degradation efficiency of fractions extracted from B. subtilis YB1 was 66.8% by EXF and 15.8% by ENF, but neither EXF nor ENF extracted from A. niger YF1 had the activity of degrading nicosulfuron.