Attrition from randomized controlled trials of pharmacological weight loss agents: a systematic review and analysis.

Clinical trials of obesity treatments have been limited by substantial dropout. Participant-level variables do not reliably predict attrition, and study-level variables have not yet been examined. We searched MEDLINE and identified 24 large randomized controlled trials of weight loss medications. These trials were comprised of 23 placebo and 32 drug groups. Two authors independently extracted the following for each treatment group: (i) treatment received; (ii) design characteristics (inclusion of a lead-in period, selection of participants with weight-related comorbidities, study location and number of study visits); (iii) sample characteristics (sample size, % female, and mean baseline age and body mass index); and (iv) attrition (total, adverse event [AE]-related and non-AE-related) at 1 year. The primary outcome was total attrition, which was significantly related to treatment (i.e. 34.9%, 28.6%, 28.3% and 35.1% in placebo, orlistat, sibutramine and rimonabant groups, respectively, P < 0.0001). In adjusted multivariable models, total attrition was significantly lower in groups that completed a pre-randomization lead-in period than in those that did not (29.1% vs. 39.9%, P < 0.01). Gender also was significantly related to total attrition; groups with more women had higher dropout (P < 0.01). The pattern was similar for predicting non-AE-related attrition. Findings suggest ways to design studies that maximize retention.

Insulin-like growth factor (IGF) system in the bovine mammary gland and milk.

Primary bovine mammary cells express the two IGF receptors (IGF-IR, IGF-IIR), insulin receptor, and four IGFBPs (IGFBP-2, -3, -4, and -5). Examination of the IGF-IR during the mammary gland lactation cycle shows that IGF-IR number declines at parturition, a change that coincides with decreases in the blood level of its ligand, IGF-I. IGF-II and IGF-IIR are largely unchanged. IGFBP-3 is the predominant mammary IGFBP and its concentration also declines in blood and milk during lactation compared to prepartum and involution periods. Time of lactation and pregnancy were the main determinants of milk but not blood IGFBP-3 levels. IGFBP-3 binds to membrane proteins of bovine mammary tissue; an IGFBP-3 binding protein has been identified as bovine lactoferrin. Lactoferrin has the capacity to compete with IGF binding to IGFBP-3. Appearance of both IGFBP-3 and lactoferrin in conditioned media of primary cultures of bovine mammary cells was stimulated by all trans retinoic acid (atRA). Furthermore, atRA was necessary for the entry of exogenously added lactoferrin into the mammary cell nucleus, while IGFBP-3 entry into the nuclei of atRA treated cells required the presence of lactoferrin. These findings reveal a novel role for lactoferrin, suggesting that lactoferrin is critically involved in the regulation of the IGF system during the involution period.

Transcriptional and posttranscriptional regulation of insulin-like growth factor binding protein-3 by cyclic adenosine 3',5'-monophosphate: messenger RNA stabilization is accompanied by decreased binding of a 42-kDa protein to a uridine-rich domain in the 3'-untranslated region.

The Madin Darby bovine kidney (MDBK) cell line was used to investigate the mechanisms underlying the cAMP regulation of insulin-like growth factor binding protein-3 (IGFBP-3) gene expression. Treatment of confluent monolayers either with forskolin or cAMP produced a 60- to 75-fold induction of IGFBP-3 mRNA and protein levels. This effect did not require new protein synthesis as inhibition of translation by cycloheximide actually caused a 2-fold increase in the cAMP induction. The rates of IGFBP-3 gene transcription, assessed by nuclear run-on assays, increased approximately 15-fold in cells exposed to cAMP. In addition, the half-life of the IGFBP-3 mRNA transcript was increased approximately 3-fold in the presence of cAMP. Gel mobility shift and competition experiments revealed the specific binding of an approximately 42-kDa cytoplasmic protein factor to the 3'-untranslated region (3'-UTR) of the IGFBP-3 mRNA. A 21-nucleotide uridine-rich segment that contained no AUUUA motif was sufficient for the specific binding. The binding activity of this protein was reduced after cAMP treatment but was increased by phosphatase treatment. In conclusion, the cAMP induction of IGFBP-3 mRNA in MDBK cells occurred at both the transcriptional and posttranscriptional levels. The IGFBP-3 mRNA stabilization in MDBK cells probably involved the phosphorylation of a member of the family of U-rich region mRNA-binding proteins and is the first reported member whose RNA-binding activity is reduced by cAMP.

Connective tissue growth factor (IGFBP-rP2) expression and regulation in cultured bovine endothelial cells.

Media from large vessel endothelial cells (pulmonary artery, aorta) contained intact connective tissue growth factor (CTGF) and a dominant 19-kDa band. N-terminal analysis of the 19-kDa band showed sequence corresponding to CTGF amino acid 181-190, suggesting that the 19-kDa band represented a proteolytic fragment of CTGF. Intact CTGF was increased by cAMP but not by transforming growth factor-beta (TGFbeta). CTGF messenger RNA (mRNA) was not changed by cAMP nor TGFbeta. In two microvessel endothelial cells, mRNA was found at low levels by PCR and Northern analysis, but no CTGF protein was seen on Western analysis. In the microvessel cells, TGFbeta increased and cAMP did not change CTGF mRNA levels, with neither TGFbeta nor cAMP increasing CTGF protein. The discordance between protein and mRNA levels in large vessel and microvessel endothelial cells was mostly explained by the effects of cAMP and TGFbeta on media proteolytic activity; in large vessel cells, cAMP inhibited degradation of CTGF, whereas in microvessel cells, TGFbeta and cAMP stimulated proteolytic activity against CTGF. We conclude that in large vessel endothelial cells, cAMP increased intact CTGF protein by inhibiting degradation of CTGF, whereas TGFbeta stimulated neither CTGF mRNA nor protein; in microvessel cells, TGFbeta increased CTGF mRNA, while both TGFbeta and cAMP stimulated CTGF degradation.

Bovine insulin-like growth factor binding protein-3: organization of the chromosomal gene and functional analysis of its promoter.

Insulin-like growth factor binding protein-3 (IGFBP-3), the major IGFBP in the circulation, is synthesized by the vascular endothelium in vivo and has been shown to be an important modulator of the physiological effects of IGF. IGFBP-3 is regulated by a number of growth factors/cytokines to which the vascular endothelium is exposed, including IGF-I stimulation and TGF-beta1 inhibition of IGFBP-3 in cultured endothelial cells. To understand the mechanisms of transcriptional regulation of IGFBP-3, we have cloned the bovine IGFBP-3 gene and begun the functional analysis of its promoter. Southern analysis indicated a single copy gene. The gene spanned approximately 10 kb and was divided into five exons, the fifth containing the 3' untranslated region. The transcription start site was 137 bp upstream of the initiation codon and a TATA box was located 26 bp 5' to this CAP site. No CAAT box was present but a GC rich sequence element, containing two overlapping putative AP-2 binding elements, was located 5' to the TATA box. Transient transfection studies with a series of 5' truncated luciferase reporter constructs were conducted in primary cultures of bovine aorta endothelial cells. Results of the transfection studies indicated that 1) nearly 80% of the maximal basal promoter activity was retained within the first 130 bp of the 5' flanking sequence; 2) this region responded to IGF-I, despite lacking the TTF-1/TTF-2 (thyroid specific transcription factors) binding elements that are required for IGF-I stimulation of thyroglobulin synthesis. These binding elements have also been suggested to be involved in IGF-I regulation of IGFBP-3 transcription, thus, implying the existence of novel cis-acting elements that mediate the IGF-I stimulation of bovine endothelial cell IGFBP-3 mRNA synthesis; 3) deletion of the GC rich sequence element resulted in a 60% reduction in basal promoter activity as well as loss of the IGF-1 stimulatory effect; 4) the TGF-beta1 mediated inhibition of IGFBP-3 transcription required sequence element(s) beyond 1.5 kb of its promoter.

Regulation of endothelial IGFBP-3 synthesis and secretion by IGF-I and TGF-beta.

We have examined the regulation of endothelial IGFBP-3 production by IGF-I and TGF-beta, two growth factors thought to play a major roles in the complications of diabetes mellitus. In addition, we developed a sensitive method for IGFBP-3 mRNA quantitation by adapting the fluorescent modification of the competitive PCR strategy. Our results using both Northern analysis and the fluorescent competitive PCR method indicate that: (1) IGFBP-3 mRNA is increased 2- to 10-fold by IGF-I and maximally reduced to 20% of control by TGF-beta; (2) the changes in mRNA levels correlate with the levels of IGFBP-3 protein secreted into the media by these cells; (3) the induction of IGFBP-3 mRNA and protein by IGF-I analogs was directly related to their ability to bind to the type I IGF receptor, reflecting an IGF-I receptor-mediated process; and (4) steady state IGFBP-3 mRNA levels did not change significantly after a 6 h incubation with actinomycin D in the presence or absence of the growth factors suggesting that the observed IGF-I/TGF-beta effects occur at the level of gene transcription rather than mRNA stability.

Differential expression of two mRNAs from a single gene encoding an HMG1-like DNA binding protein of African trypanosomes.

We have isolated cDNA clones expressing a member of the high mobility group (HMG) protein family by screening a Trypanosoma brucei rhodesiense expression cDNA library with multimerized oligonucleotides corresponding to an octamer transcriptional regulatory sequence motif. The trypanosome DNA binding protein (TDP-1) encoded by these cDNAs contains two domains that show striking sequence similarity to the consensus sequence for HMG1-like DNA binding domains (HMG boxes). Southern blot analysis is consistent with TDP-1 being encoded by a single copy gene. The cDNA clones are derived from 2 mature mRNA species of approximately 1.6 and 2.3 kb in length that are generated by differential polyadenylation at sites 563 nucleotides and 1113 nucleotides downstream from the stop codon. Stage specific differences exist in the steady state levels of the 2 mRNAs: bloodstream parasites contain predominantly the 1.6-kb mRNA, while procyclic culture forms express predominantly the 2.3-kb mRNA.

Characterization of trypanosome protein phosphatase 1 and 2A catalytic subunits.

Oligonucleotides corresponding to highly conserved regions of mammalian protein phosphatase catalytic subunits were used in the polymerase chain reaction (PCR) to generate an amplification product from genomic DNA of Trypanosoma brucei rhodesiense. The PCR product was used to screen a T. b. rhodesiense cDNA library for cDNA clones encoding putative protein phosphatase catalytic subunits. Two cDNA clones, (TPP1A and TPP1B) representing two distinct type 1 catalytic subunit isotypes, encode 39-kDa proteins of 346 amino acids that show 66% and 40% identity, respectively, to mammalian protein phosphatase 1 and 2A catalytic subunits. Both cDNAs are derived from 2.3-kb mRNAs, and Northern blot analysis has provided indirect evidence that these mRNAs are part of the same transcription unit as mRNAs for RNA polymerase II largest subunit. Another cDNA, TPP2, represents the type 2A class of phosphatases and codes for a 34.5-kDa protein of 303 amino acids. The deduced amino acid sequence has 39% and 55% identity, respectively, to the catalytic subunits of mammalian protein phosphatase 1 and 2A. Southern and Northern blot analyses are consistent with TPP2 being encoded by a single copy gene from which is derived a mRNA of 2.5 kb. This finding constitutes the first example in eukaryotes in which a single gene encodes the type 2A class of protein phosphatases. Sera from mice immunized with TPP1A fusion protein reacted with the catalytic subunits of mammalian types 1, 2A and 2B protein phosphatases. However, antisera to TPP2 fusion protein was specific for the type 2A catalytic subunit and recognized a polypeptide of 35 kDa in a Western blot of crude trypanosomal lysate.

Characterization of a myosin-like antigen from Onchocerca volvulus.

Recombinant cDNAs expressing an immunodominant antigen (Onchoag-1) of Onchocerca volvulus were identified by immunoscreening a cDNA expression library. The Onchoag-1 cDNAs are derived from an 8-kb mRNA that codes for a protein with an apparent molecular mass of 200 kDa. Indirect immunofluorescence using antisera against a recombinant fusion protein showed that Onchoag-1 is located in the muscle tissues of adult O. volvulus. The 2-kb sequence of one of the cDNAs contains a single open translation reading frame that encodes a protein with sequence similarities to Caenorhabditis elegans myosin heavy chain. Analysis of the 3' region of Onchoag-1 chromosomal gene reveals that it is frequently interrupted by short introns that follow the GT/AG rule at their splice sites. Studies on this myofibrillar antigen should contribute to our understanding of muscle function in O. volvulus as well as provide useful insight to the genesis of the immunopathological damage that is often associated with allergic reactions (the Mazzotti reactions) in onchocerciasis patients, following the administration of a chemotherapeutic agent such as diethylcarbamazine.

Construction of Onchocerca volvulus cDNA libraries and partial characterization of the cDNA for a major antigen.

Adult Onchocerca volvulus were isolated from nodules removed from onchocerciasis patients at four locations--two in the West African Sudan-savanna region (near Bamako, Mali, and Touboro, Cameroon), one in a West African forest region (Kumba, Cameroon) and one near Guatemala City, Guatemala. Four different cDNA expression libraries were constructed in bacteriophage lambda gt11 using poly(A)+ RNA from the adult female worms. Individual cDNA clones of single copy genes were used to compare the genomes of parasites from the different locales and to show that the haploid genome of O. volvulus is 1.5 x 10(8) base pairs. About 1 in 700 recombinant clones in each of the four amplified cDNA libraries produces a fusion protein recognized by pooled human anti-O. volvulus antisera. Partial sequence determination of a 2.0 kb cDNA clone for an O. volvulus protein that induces an immunodominant response in rabbits revealed that this antigen has sequence similarities with Caenorhabditis elegans myosin and with schistosome paramyosin (which confers partial protection against schistosome infection). The four cDNA libraries have been deposited with American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852, U.S.A., for general distribution under ATCC Number 37509.

Regional distribution of type II Ca2+/calmodulin-dependent protein kinase in rat brain.

The distribution of type II Ca2+/calmodulin-dependent protein kinase has been mapped in rat brain by immunochemical and immunohistochemical methods using an antibody against its alpha-subunit. The concentration of the kinase, measured by radioimmunoassay, varies markedly in different brain regions. It is most highly concentrated in the telencephalon where it comprises approximately 2% of the total hippocampal protein, 1.3% of cortical protein, and 0.7% of striatal protein. It is less concentrated in lower brain structures, ranging from about 0.3% of hypothalamic protein to 0.1% of protein in the pons/medulla. The gradient of staining intensity observed in brain sections by immunohistochemistry corroborates this distribution. Neurons and neuropil of the hippocampus are densely stained, whereas little staining is observed in lower brain regions such as the superior colliculus. Within the diencephalon and midbrain, dense staining is observed only in thalamic nuclei and the substantia nigra. The skewed distribution of alpha-subunit appears to be due in part to the occurrence in the cerebellum and pons/medulla of forms of the kinase with a high ratio of beta- to alpha-subunits. However, most of the variation is due to the extremely high concentration of the kinase in particular neurons, especially those of the hippocampus, cortex and striatum. The unusually high expression of the kinase in these neurons is likely to confer upon them specialized responses to calcium ion that are different from those of neurons in lower brain regions.

Biochemical and immunochemical evidence that the "major postsynaptic density protein" is a subunit of a calmodulin-dependent protein kinase.

By three criteria, two biochemical and one immunochemical, the major postsynaptic density protein (mPSDp) is indistinguishable from the 50-kilodalton (kDa) alpha subunit of a brain calmodulin-dependent protein kinase. First, the two proteins comigrate on NaDodSO4/polyacrylamide gels. Second, iodinated tryptic peptide maps of the two are identical. Finally, a monoclonal antibody (6G9) that was raised against the protein kinase binds on immunoblots to a single 50 kDa band in crude brain homogenates and to both the alpha subunit of the purified kinase and the mPSDp from postsynaptic density fractions. The purified kinase holoenzyme also contains a 60-kDa subunit termed beta. A comparison of the peptide map of beta with the maps of 60-kDa proteins from the postsynaptic density fraction suggests that beta is present there but is not the only protein present in this molecular weight range. These results indicate that the calmodulin-dependent protein kinase is a major constituent of the postsynaptic density fraction and thus may be a component of type I postsynaptic densities.

Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain.

A calcium and calmodulin-dependent protein kinase has been purified from rat brain. It was monitored during the purification by its ability to phosphorylate the synaptic vesicle-associated protein, synapsin I. A 300-fold purification was sufficient to produce kinase that is 90-95% pure as determined by scans of stained sodium dodecyl sulfate-polyacrylamide gels and has a specific activity of 2.9 mumol of 32P transferred per min/mg of protein. Thus, the kinase is a relatively abundant brain enzyme, perhaps comprising as much as 0.3% of the total brain protein. The Stokes radius (95 A) and sedimentation coefficient (16.4 S) of the kinase indicate a holoenzyme molecular weight of approximately 650,000. The holoenzyme is composed of three subunits as judged by their co-migration with kinase activity during the purification steps and co-precipitation with kinase activity by a specific anti-kinase monoclonal antibody. The three subunits have molecular weights of 50,000, 58,000, and 60,000, and have been termed alpha, beta', and beta, respectively. The alpha- and beta-subunits are distinct peptides, however, beta' may have been generated from beta by proteolysis. All three of these subunits bind calmodulin in the presence of calcium and are autophosphorylated under conditions in which the kinase is active. The subunits are present in a ratio of about 3 alpha-subunits to 1 beta/beta'-subunit. We therefore postulate that the 650,000-Da holoenzyme consists of approximately 9 alpha-subunits and 3 beta/beta'-subunits. The abundance of this calmodulin-dependent protein kinase indicates that its activation is likely to be an important biochemical response to increases in calcium ion concentration in neuronal tissue.