Percutaneous Closure of Bilateral Pulmonary Artery Aneurysms in Behcet's Disease.

Behcet's disease is a multisystemic vasculitis. It can affect the pulmonary artery in 2% to 5% cases. We discuss a case of a young male diagnosed with Behcet's disease on immunosuppressive therapy who presented with bilateral pulmonary artery aneurysms which were closed with covered stent and other devices.

Retroviral vector integration in post-transplant hematopoiesis in mice conditioned with either submyeloablative or ablative irradiation.

X-linked chronic granulomatous disease (X-CGD) is an inherited immunodeficiency with absent phagocyte NADPH-oxidase activity caused by defects in the gene-encoding gp91(phox). Here, we evaluated strategies for less intensive conditioning for gene therapy of genetic blood disorders without selective advantage for gene correction, such as might be used in a human X-CGD protocol. We compared submyeloablative with ablative irradiation as conditioning in murine X-CGD, examining engraftment, oxidase activity and vector integration in mice transplanted with marrow transduced with a gamma-retroviral vector for gp91(phox) expression. The frequency of oxidase-positive neutrophils in the donor population was unexpectedly higher in many 300 cGy-conditioned mice compared with lethally irradiated recipients, as was the fraction of vector-marked donor secondary CFU-S12. Vector integration sites in marrow, spleen and secondary CFU-S12 DNA from primary recipients were enriched for cancer-associated genes, including Evi1, and integrations in or near cancer-associated genes were more frequent in marrow and secondary CFU-S12 from 300 cGy-conditioned mice compared with fully ablated mice. These findings support the concept that vector integration can confer a selection bias, and suggest that the intensity of the conditioning regimen may further influence the effects of vector integration on clonal selection in post-transplant engraftment and hematopoiesis.

Effect of fenvalerate technical grade on acetyl cholinesterase activity in Indian bullfrog Haplobatrachus tigerinus (Daudin).

Indian bullfrog Haplobatrachus tigerinus (Daudin) was exposed to sublethal dose (1/3 of LC50 I.E. 1.166 mg/kg) of fenvalerate technical grade and the effect was studied on the specific activity of acetyl cholinesterase in the different tissues of frog viz., brain, muscle, liver, kidney and testis at different time periods viz., 3,6, 12, 24, 48 and 72 hours. The inhibition of specific activity of acetyl cholinesterase was in the order of kidney > brain > muscle > liver > testis. A significant inhibition was noticed in kidney at 12 hours (-64.33%) and no effect was noticed at 3 hours in testis (+0.67%). The AChE activity was inhibited in first three hours of administration of fenvalerate in all the tissue tested. The inhibition continued upto 6 hours or 2 hours in different tissue but the recovery was started by 24 hours and almost completed by 72 hours.

Bioaccumulation of fenvalerate technical grade in different organs of the frog Haplobatrachus tigerinus (Daudin).

Bioaccumulation studies of fenvalerate were conducted on Indian bullfrog Haplobatrachus tigrinus (Daudin) after exposure to sublethal intraperitoneal dose of technical grade fenvalerate (1/3 LD50 i.e. 116.66 microg/kg body weight) at 3, 6, 12, 24, 48 and 72 hours schedule. The tissues viz., muscle, liver, kidney, testis, brain, and whole body accumulation was analysed for residue estimations. In all the tissues, analysed maximum residue was recovered in the initial stages of exposure (3 and 6 hours). However, in brain the residues remained up to 72 hours. In the whole body, analysis after 3 hours of exposure 78.65% residue was recovered and by the time 72 hours passed only, 9.4% residue was recovered. The decline in residue levels along with the period of exposure indicates the fast acting nature of fenvalerate and metabolites.

Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods.

To better characterize lentiviral vector supernatants, we compared three methods of titer assessment. These titer methods include assessment of vector RNA sequences in supernatants, DNA sequences in transduced cells, and vector expression in transduced cells (using a vector which expressed the green fluorescence protein, GFP). For analysis of RNA and DNA, we developed a real-time PCR method for detecting the lentiviral packaging sequence and used this methodology to quantitate the number of vector sequences. Vector expression was assessed by flow cytometric analysis for GFP. As functional titers (DNA and GFP expression titers) are dependent on transduction efficiency, we calculated the titer of a lentiviral vector, RRL-CMV-GFP, after transduction of 293, HeLa, or Mus dunni cells. Genomic DNA was extracted at 4 and 14 days after transduction and the number of vector DNA molecules was determined against a plasmid standard. Of the three cell lines tested, 293 cells provided the highest rate of transduction (PCR estimated DNA titer for RRL-CMV-GFP vector was 2.52 +/- 0.25 x 10(6) molecules/ml at 14 days, and 2.31 +/- 0.15 x 10(6) molecules/ml at 4 days). When titer was calculated based on GFP expression, the highest titer was also obtained on 293 cells (0.26 +/- 0.04 x 10(6) TU/ml at 14 days, and 0.24 +/- 0.03 +/- 10(6) TU/ml at 4 days). The titers obtained by GFP expression assay were approximately one log lower than those obtained by DNA analysis suggesting that variability in vector expression may underestimate titer. Measurement of RNA titers directly from vector supernatants against a plasmid standard indicated that the RNA titers are substantially higher than the DNA (approximately 10(3)-fold) and GFP titers (approximately 10(4)-fold). To show that the lentiviral probe and primers could be used for titering a variety of lentiviral vectors, we have also used the real-time PCR method to determine the DNA titers of two other HIV1 derived vectors, RRL-PGK-GFP (6.1 +/- 1.4 x 10(5) molecules/ml), and SMPU-RRE-BN (1.26 +/- 0.2 x 10(6) molecules/ml). We conclude that of the three methods tested, titers assessed by DNA analysis of transduced cells provide the most reliable estimate of functional titers as these are least likely to be influenced by factors, such as defective interfering particles and vector expression levels. The real-time PCR method described offers a reproducible method for lentiviral titering and can be applied to a wide variety of vectors, regardless of transgene.

Benign glandular and squamous metaplastic-like cells seen in vaginal Pap smears of post hysterectomy patients: incidence and patient profile.

We have observed benign glandular cells and squamous metaplastic-like cells in vaginal Pap smears of post hysterectomy patients (PHP). Vaginal Pap smears from 1,547 PHP were retrieved. In 2% of these smears (Group A) glandular cells were observed, with the majority of the smears revealing squamous metaplastic-like cells (47%). Mucinous endocervical columnar-like cells were seen in 9% of the cases, glandular cells not resembling endocervical cells in 13%, and a combination of the former two categories in 31%. Group A patients were compared with other PHP without these cells in their vaginal smears (Group B). Several clinical and surgical parameters were evaluated. A distinctive clinical profile was not identified for either group of patients (A or B). Of patients in group A 49.8% had a history of a previous gynecologic malignancy (Group B: 19%). Based on our study, we postulate that in the absence of a clinically identifiable source of these cells, the most likely source of origin is probably vaginal adenosis not associated with DES exposure in utero or a metaplastic phenomenon perhaps related to therapy. These cells do not seem to be related to imminent neoplasia or dysplasia.

Nonphosphorylated peptide ligands for the Grb2 Src homology 2 domain.

Critical intracellular signals in normal and malignant cells are transmitted by the adaptor protein Grb2 by means of its Src homology 2 (SH2) domain, which binds to phosphotyrosyl (pTyr) residues generated by the activation of tyrosine kinases. To understand this important control point and to design inhibitors, previous investigations have focused on the molecular mechanisms by which the Grb2 SH2 domain selectively binds pTyr containing peptides. In the current study, we demonstrate that the Grb2 SH2 domain can also bind in a pTyr independent manner. Using phage display, an 11-amino acid cyclic peptide, G1, has been identified that binds to the Grb2 SH2 domain but not the src SH2 domain. Synthetic G1 peptide blocks Grb2 SH2 domain association (IC50 10-25 microM) with a 9-amino acid pTyr-containing peptide derived from the SHC protein (pTyr317). These data and amino acid substitution analysis indicate that G1 interacts in the phosphopeptide binding site. G1 peptide requires a YXN sequence similar to that found in natural pTyr-containing ligands, and phosphorylation of the tyrosine increases G1 inhibitory activity. G1 also requires an internal disulfide bond to maintain the active binding conformation. Since the G1 peptide does not contain pTyr, it defines a new type of SH2 domain binding motif that may advance the design of Grb2 antagonists.

RecA protein assisted selection reveals a low fidelity of recognition of homology in a duplex DNA by an oligonucleotide.

We have developed an in vitro selection procedure to elucidate the specificity of RecA assisted oligonucleotide recognition of double stranded DNA. The procedure was based on formation of a synaptic complex between an oligonucleotide-RecA filament and a supercoiled plasmid bearing a homologous partially degenerate region. The specificity of the selection depended on the reaction conditions: starting with a population that had, on average, 2.8 randomly distributed mismatches out of 27 bp, a population selected in the presence of 100 mM KCl had on average 1.0 mismatches, while a population selected at low ionic strength was less specific and had, on average, 2.0 mismatches. From the distributions of mismatches observed we calculated that the average destabilization free energy for one mismatch is 1.7(+/-0.5) kcal/mol. This is substantially less than the free energy for the incorporation of one mismatch in naked DNA duplex or a Py-Pu-Py triplex. Thus, RecA has an ability to decrease the fidelity of the homologous pairing reaction and minimize the cost of pairing between similar but not identical sequences. This "antiproofreading" activity of RecA protein does not require ATP hydrolysis.

Multiple Grb2-protein complexes in human cancer cells.

Grb2 is an SH2/SH3 domain-containing adaptor protein that links receptor tyrosine kinases to the ras signaling pathway. The Grb2-SH2 domain binds phosphotyrosine sequences on activated tyrosine kinases, and one target of the SH3 domains is the ras-nucleotide-exchange factor Sos1. We have examined Grb2-protein interactions in human cancer cells that over-express the receptor tyrosine kinase erbB2. Our results show that the 2 Grb2-SH3 domains complex with Sos1, dynamin and at least 4 other proteins (p228, p140, p55, p28) in these cells. The 2 Grb2-SH3 domains bind these proteins differently, with the N-terminal SH3 domain interacting preferentially with p228, Sos1, p140 and dynamin. The C-terminal SH3 domain has higher affinity toward p28. The Grb2-SH3 domain interactions appear to be similar in erbB2 over-expressing breast, ovarian and lung cancer cells. Also, the major tyrosine-phosphorylated proteins that associate with Grb2 in erbB2 over-expressing cancer cells appear to be erbB2 and Shc. The multiple Grb2-SH3 domain interactions in these cells may mediate novel cellular functions.

Quantitative analysis of Grb2-Sos1 interaction: the N-terminal SH3 domain of Grb2 mediates affinity.

Grb2 is an adaptor protein that links receptor and cytoplasmic tyrosine kinases to the Ras signalling pathway by binding the Ras-specific guanine nucleotide exchange factor, Sos1, through its SH3 domains. The Grb2-SH3 domain binding has been localized to the carboxy-terminal two hundred amino acids of Sos1 (Sos1-c). By using real time biospecific interaction analysis (BIAcore), we studied the kinetic parameters and binding affinity of the Grb2-Sos1-c interaction. The binding of Grb2 to Sos1-c is a high affinity interaction with a moderate association rate (9.45 x 10(4) per M per s), a slow dissociation rate (13.8 x 10(-5) s), and an affinity constant of 1.48 nM. BIAcore measurements on isolated N-terminal and C-terminal SH3 domains (NSH3 and CSH3) further indicate that the high affinity Grb2-Sos1-c interaction is primarily mediated through the NSH3 domain (Kd = 1.68 nM). The CSH3 domain shows substantially reduced binding to Sos1-c in these measurements. Inhibition studies with BIAcore using proline rich peptides derived from the C-terminus of Sos1 show that there is a single major binding site for Grb2 in Sos1. This binding site is contained within the peptide N20, which corresponds to amino acids 1143-1162 of Sos1. This peptide completely blocks the Grb2-Sos1-c and NSH3-Sos1-c interactions with IC50 values of 8 microM and 4 microM respectively. The discrete interaction between the NSH3 domain and the N20 peptide may be amenable for drug discovery through screening or peptidomimetic approaches.

Photoaffinity labeling of rhodopsin and bacteriorhodopsin.

Photoaffinity labeling with bovine rhodopsin using a retinal with a fixed 11-cis-ene cross-linked exclusively to Trp-265/Leu-266 in helix F, showing that the beta-ionone C-3 is close to helix F. Moreover, since these labeled amino acids are in the middle of helix F, while the Schiff-base linkage to Lys-296 at the other terminus of the chromophore is also in the middle of helix G, the chromophore lies horizontally near the center of the lipid bilayer. In bacteriorhodopsin, photoaffinity studies using a retinal with a C-10 tritiated phenylazide appended through a 13 A spacer cross-linked to Arg-175/Asn-176 on the cytoplasmic side of helix F; this indicates that 9-Me points toward the extracellular space. This result agrees with our earlier studies with 9-sulfate analogs but is opposite to that deduced by biophysical measurements.

Screening combinatorial antibody libraries for catalytic acyl transfer reactions.

A bacteriophage lambda vector system for the expression of Fab fragments from the mouse antibody repertoire in Escherichia coli has been described. We have used this system to generate a catalytic antibody from a combinatorial antibody library. Monoclonal antibody 43C9 was raised against a transition state analogue of the hydrolysis of carboxyamide. mRNA from hybridoma cells expressing this antibody was cloned into phage lambda and clones that expressed the mRNA for either the heavy or the light chain of the antibody were isolated. These individual libraries were then crossed to generate a combinatorial library in which clones coexpressed the heavy and light chains. This library was screened for antibodies/Fab fragments that bound to the original antigen with high affinity. DNA sequencing showed that these fragments were the same as those in antibody 43C9. Three different clones were found to catalyse the hydrolysis of carboxyamide. More efficient expression vectors and improved screening techniques should lead to the isolation of many more catalytic antibodies from combinatorial antibody libraries.

Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda.

A novel bacteriophage lambda vector system was used to express in Escherichia coli a combinatorial library of Fab fragments of the mouse antibody repertoire. The system allows rapid and easy identification of monoclonal Fab fragments in a form suitable for genetic manipulation. It was possible to generate, in 2 weeks, large numbers of monoclonal Fab fragments against a transition state analog hapten. The methods described may supersede present-day hybridoma technology and facilitate the production of catalytic and other antibodies.

Cloning of the immunological repertoire in Escherichia coli for generation of monoclonal catalytic antibodies: construction of a heavy chain variable region-specific cDNA library.

Efficient generation of catalytic antibodies is uniquely dependent on the exact nature of the binding interactions in the antigen-antibody complex. Current methods for generation of monoclonal antibodies do not efficiently survey the immunological repertoire and, therefore, they limit the number of catalysts that can be obtained. We are exploring methods to clone and express the immunological repertoire in Escherichia coli. As the essential first step, we present here a method for the establishment of a highly diverse heavy chain variable region library. Consequently, it should now be possible to express and recombine the heavy and light chain variable region fragments to generate a large array of functional combining portions of the antibody molecule. This technology may provide an alternative to the hybridoma methodology for accessing the monoclonal antibody specificity of the immune system.