Cytotoxic Tumor-Targeting Peptides From In Vivo Phage Display.

We previously utilized an in vivo peptide phage display selection technique, which included the use of detergent elution of phage from excised tumor, to obtain tumor-targeting phage with the ability to extravasate the vasculature and bind directly to prostate tumor tissue. It is hypothesized that this same in vivo phage selection technique can be used to functionally select for molecules that not only bind to cancer cells but also kill them. Here we analyzed two different in vivo phage display selected phage clones, G1 and H5, retrieved from PC-3 human prostate carcinoma xenografted tumors.

Relations between rate of negative reinforcement and the persistence of task completion.

Research has shown that differential reinforcement of alternative behavior (DRA) can be an effective intervention to address problem behavior maintained by negative reinforcement emitted by young children. However, few studies have evaluated the variables that are related to long-term maintenance (i.e.

A high throughput screening assay system for the identification of small molecule inhibitors of gsp.

Mis-sense mutations in the α-subunit of the G-protein, Gsα, cause fibrous dysplasia of bone/McCune-Albright syndrome. The biochemical outcome of these mutations is constitutively active Gsα and increased levels of cAMP. The aim of this study was to develop an assay system that would allow the identification of small molecule inhibitors specific for the mutant Gsα protein, the so-called gsp oncogene.

Genomics in clinical practice: lessons from the front lines.

The price of whole-genome and -exome sequencing has fallen to the point where these methods can be applied to clinical medicine. Here, we outline the lessons we have learned in converting a sequencing laboratory designed for research into a fully functional clinical program.

Quantitative Analysis of Guanine Nucleotide Exchange Factors (GEFs) as Enzymes.

The proteins that possess guanine nucleotide exchange factor (GEF) activity, which include about ~800 G protein coupled receptors (GPCRs), 15 Arf GEFs, 81 Rho GEFs, 8 Ras GEFs, and others for other families of GTPases, catalyze the exchange of GTP for GDP on all regulatory guanine nucleotide binding proteins. Despite their importance as catalysts, relatively few exchange factors (we are aware of only eight for ras superfamily members) have been rigorously characterized kinetically. In some cases, kinetic analysis has been simplistic leading to erroneous conclusions about mechanism (as discussed in a recent review).

Nucleotide exchange factors: Kinetic analyses and the rationale for studying kinetics of GEFs.

Exchange factors are enzymes that catalyze the exchange of GTP for GDP on guanine nucleotide binding proteins. Progress in understanding the molecular basis of action and the cellular functions of these enzymes has largely come from structural determinations (e.g.

D2-like dopamine and β-adrenergic receptors form a signaling complex that integrates Gs- and Gi-mediated regulation of adenylyl cyclase.

β-Adrenergic receptors (βAR) and D(2)-like dopamine receptors (which include D(2)-, D(3)- and D(4)-dopamine receptors) activate G(s) and G(i), the stimulatory and inhibitory heterotrimeric G proteins, respectively, which in turn regulate the activity of adenylyl cyclase (AC). β(2)-Adrenergic receptors (β(2)AR) and D(4)-dopamine receptors (D(4)DR) co-immunoprecipitated when co-expressed in HEK 293 cells, suggesting the existence of a signaling complex containing both receptors. In order to determine if these receptors are closely associated with each other, and with other components involved in G protein-mediated signal transduction, β(2)AR, D(4)DR, G protein subunits (Gα(i1) and the Gβ(1)γ(2) heterodimer) and AC were tagged so that bioluminescence resonance energy transfer (BRET) could be used to monitor their interactions.

Modulation of the interaction between neurotensin receptor NTS1 and Gq protein by lipid.

Membrane lipids have been implicated to influence the activity of G-protein-coupled receptors (GPCRs). Almost all of our knowledge on the role of lipids on GPCR and G protein function comes from work on the visual pigment rhodopsin and its G protein transducin, which reside in a highly specialized membrane environment. Thus, insight gained from rhodopsin signaling may not be simply translated to other nonvisual GPCRs.

Large putative PEST-like sequence motif at the carboxyl tail of human calcium receptor directs lysosomal degradation and regulates cell surface receptor level.

A deletion between amino acid residues Ser(895) and Val(1075) in the carboxyl terminus of the human calcium receptor (hCaR), which causes autosomal dominant hypocalcemia, showed enhanced signaling activity and increased cell surface expression in HEK293 cells (Lienhardt, A., Garabédian, M. G.

An autoinhibitory helix in the C-terminal region of phospholipase C-β mediates Gαq activation.

The enzyme phospholipase C-β (PLCβ) is a crucial regulator of intracellular calcium levels whose activity is controlled by heptahelical receptors that couple to members of the Gq family of heterotrimeric G proteins. We have determined atomic structures of two invertebrate homologs of PLCβ (PLC21) from cephalopod retina and identified a helix from the C-terminal regulatory region that interacts with a conserved surface of the catalytic core of the enzyme. Mutations designed to disrupt the analogous interaction in human PLCβ3 considerably increase basal activity and diminish stimulation by Gαq.

A complex chromosome rearrangement, der(6)ins(6)(p21.1q25.3q27)inv(6)(p25.3q27), in a child with cleidocranial dysplasia.

Complex chromosome rearrangements (CCRs) are structural abnormalities involving >2 chromosomes or >3 breakpoints. It has been suggested that the probability of imbalance increases as the number of breakpoints increase. Here we report a 7-month-old, Hispanic girl presenting with cleidocranial dysplasia (CCD) who was found to have a complex chromosome rearrangement of chromosome 6.

Gipc3 mutations associated with audiogenic seizures and sensorineural hearing loss in mouse and human.

Sensorineural hearing loss affects the quality of life and communication of millions of people, but the underlying molecular mechanisms remain elusive. Here, we identify mutations in Gipc3 underlying progressive sensorineural hearing loss (age-related hearing loss 5, ahl5) and audiogenic seizures (juvenile audiogenic monogenic seizure 1, jams1) in mice and autosomal recessive deafness DFNB15 and DFNB95 in humans. Gipc3 localizes to inner ear sensory hair cells and spiral ganglion.

Rab1 small GTP-binding protein regulates cell surface trafficking of the human calcium-sensing receptor.

The human calcium-sensing receptor (hCaR) is a family-3/C G-protein-coupled receptor that regulates Ca(2+) homeostasis by controlling parathyroid hormone secretion. Here we investigated the role of Rab1, a small GTP-binding protein that specifically regulates protein transport from the endoplasmic reticulum to the Golgi, in cell surface transport of the hCaR. Cell surface expression of hCaR transiently expressed in human embryonic kidney 293 cells was strongly augmented by coexpression of Rab1 and attenuated by disruption of endogenous Rab1 function by expression of the dominant-negative Rab1N124I mutant or depletion of Rab1 with small interfering RNA.

Pericentric inversion, inv(14)(p11.2q22.3), in a 9-month old with features of Goldenhar syndrome.

Goldenhar syndrome, also called hemifacial microsomia or oculo-auriculo-verterbal dysplasia (OAVS) (MIM 164210), is a birth defect involving the first and second branchial arch derivatives with an incidence of 1/5000. The variable phenotype includes mostly unilateral deformity of the external ear and small ipsilateral half of the face with epibulbar dermoid and vertebral anomalies. A genome-wide search in one family suggested linkage to a region of 10.

Sar1-dependent trafficking of the human calcium receptor to the cell surface.

The molecular mechanisms underlying the exit from the endoplasmic reticulum (ER) for cell surface trafficking of the human calcium receptor (hCaR) remain poorly understood. We investigated the role of the Sar1 small GTP-binding protein in cell surface transport of the hCaR. Disruptions of endogenous Sar1 function with the constitutively active Sar1H79G mutant or depletion using small interfering RNA, attenuates cell surface expression of the hCaR.

Molecular cytogenetic characterization of an interstitial de novo 13q deletion in a 3-month-old with severe pediatric gastroesophageal reflux.

Gastroesophageal reflux (GER) occurs when gastric contents travel back into the esophagus through the esophageal sphincter. GER is very common in infants with most growing out of it, but some continue to have chronic symptoms throughout childhood and adulthood. A gene for severe pediatric gastroesophageal reflux disease (GERD) was identified by linkage analysis and was mapped to chromosome 13.

A preliminary analysis of instructional control in the maintenance of appropriate behavior.

This bridge study evaluated the effects of contingency-specifying instructions (CSIs) and incomplete instructions (IIs) in terms of establishing instructional control of appropriate behavior. Results suggested that instructional control and maintenance were achieved with CSIs but not with IIs. Results are discussed in terms of the potential use of instructional control in the maintenance of appropriate behavior for children with attention deficit hyperactivity disorder.

Laparoscopic gastric banding in a kidney-pancreas transplant recipient with new onset type II diabetes mellitus associated with morbid obesity.

Combined kidney-pancreas transplantation is the treatment of choice for end-stage diabetic nephropathy. Post-transplant weight gain increases the risk for post-transplant complications and death owing to cardiovascular events. Gastric banding is an established treatment for moderate morbid obesity.

An evaluation of stimulant medication on the reinforcing effects of play.

Although a vast literature has indicated that stimulant medications are effective for reducing inappropriate behavior in children with attention deficit hyperactivity disorder (ADHD), the effects of stimulant medication on ancillary behaviors (e.g., play) have yet to be investigated with the same rigor.

Role of chromosome 1 pericentric heterochromatin (1q) in pathogenesis of myelodysplastic syndromes: report of 2 new cases.

Chromosome 1 pericentromeric heterochromatin (1q) has been shown to play an important role in the pathogenesis of non-Hodgkin lymphoma and multiple myeloma. Myelodysplastic syndrome (MDS) results from marrow failure in two or more cell lineages. Although trisomy 1q has been reported in MDS, it is usually present with additional common abnormalities such as trisomy 8, monosomy 5 or monosomy 7, leading to speculation that 1q abnormalities are mostly secondary events representing clonal evolution.

Molecular cytogenetic characterization of a unique and complex de novo 8p rearrangement.

Human chromosome 8p is prone to recurrent rearrangements with inv dup del(8p) being most common. Each of these recurrent rearrangements is associated with different clinical manifestations. Some of these recurrent rearrangements at 8p are mediated by an 8p submicroscopic paracentric inversion between the olfactory gene clusters present in one of the parents.

Prolonged preleukemic phase of chronic myelogenous leukemia.

We report the detailed features of a unique case of a 33-year-old male in whom a large percentage of marrow (68%) and blood (31%) cells carried the Philadelphia chromosome by cytogenetics and FISH, as well as the p210 BCR-ABL transcript by RT-PCR, for 15 months prior to development of chronic myelogenous leukemia. The patient was closely followed throughout the preleukemic period with repeat blood and bone marrow analysis with no hematologic or pathologic evidence of leukemia, despite harboring a large number (65%) of Ph+ marrow cells. we report the detailed history of a 33-year-old male who carried a large number of BCR-ABL-positive cells in the marrow and blood for more than 15 months before finally presenting with classic chronic myelogenous leukemia.

Dimerization of the class A G protein-coupled neurotensin receptor NTS1 alters G protein interaction.

G protein-coupled receptors (GPCRs) have been found as monomers but also as dimers or higher-order oligomers in cells. The relevance of the monomeric or dimeric receptor state for G protein activation is currently under debate for class A rhodopsin-like GPCRs. Clarification of this issue requires the availability of well defined receptor preparations as monomers or dimers and an assessment of their ligand-binding and G protein-coupling properties.