Translation of the human angiotensin II type 1 receptor mRNA is mediated by a highly efficient internal ribosome entry site

Activation of the angiotensin II type 1 receptor (AT1R) is closely involved in the pathogenesis of cardiovascular disease. The human AT1R (hAT1R) mRNA splice variants have long 5'-untranslated regions (5'-UTRs) ranging from 272 to 414 bp that have the potential to form stable secondary structures. In this study, we show that the 5'-UTR of hAT(1)R mRNAs contains an internal ribosome entry site (IRES) located within the first 40 bp of the proximal end of exon 1. Experiments utilizing the hAT1R 5'-UTR as a molecular decoy demonstrate a reduction in IRES activity of approximately 50%. This inhibition is most efficient for the hAT1R IRES suggesting that a defined set of trans-factors are required to initiate translation through this cis-element. Translation initiation from the hAT1R IRES appears to be physiologically relevant since IRES activity was maintained during serum starvation, a cellular stress known to inhibit cap-dependent translation. These results suggest that cap-independent translation initiation by internal ribosome entry may represent an important mechanism for the regulation of hAT1R expression.     

Effect of acute stress on oesophageal motility in patients with gastro-oesophageal reflux disease.

BACKGROUND AND AIM--Sixty four per cent of people with heartburn believe that it is exacerbated by stress. An alteration in oesophageal motility is one possible mechanism for this apparent change with stress. This study aimed to assess the effect of acute stressors on oesophageal motility in patients with gastro-oesophageal reflux disease (GORD). METHODS--Sixty patients were studied. Twenty had oesophagitis, 20 had increased oesophageal acid exposure on pH monitoring but no endoscopic oesophagitis, and 20 had neither oesophagitis nor abnormal oesophageal acid exposure. Oesophageal motility was studied in these patients during psychological stress (Stroop test) and physical stress (cold pressor test). RESULTS--Blood pressure (BP) and heart rate increased in response to both stressors (mean systolic BP increased by > 10 mm Hg, diastolic BP by > 4 mm Hg and heart rate by > 3 beats per minute (p < 0.00001). The amplitude, duration, and velocity of propagation of oesophageal peristaltic contractions were not altered by the stressors. The percentage of simultaneous waves increased in patients with oesophagitis during the cold pressor test (median increase in these patients was 6% (p < 0.05)). This effect was not noted in the patients without oesophagitis. CONCLUSION--Acute stressors did not induce significant changes in oesophageal motility in patients with GORD but no oesophagitis. For these patients, dysmotility is not likely to be a cause of oesophageal symptoms which are exacerbated by stress. There was, however, a significant increase in simultaneous waves during cold pressor stress in patients with oesophagitis.     

Embedding and Sustaining a Focus on Function in Specialty Research and Care

Function and the independent performance of daily activities are of critical importance to older adults. Although function was once a domain of interest primarily limited to geriatricians, transdisciplinary research has demonstrated its value across the spectrum of medical and surgical care. Nonetheless, integrating a functional perspective into medical and surgical therapeutics has yet to be implemented consistently into clinical practice. This article summarizes the presentations and discussions from a workshop, “Embedding/Sustaining a Focus on Function in Specialty Research and Care,” held on January 31 to February 1, 2019. The third in a series supported by the National Institute on Aging and the John A. Hartford Foundation, the workshop aimed to identify scientific gaps and recommend research strategies to advance the implementation of function in care of older adults. Transdisciplinary leaders discussed implementation of mobility programs and functional assessments, including comprehensive geriatric assessment; integrating cognitive and sensory functional assessments; the role of culture, environment, and community in incorporating function into research; innovative methods to better identify functional limitations, techniques, and interventions to facilitate functional gains; and the role of the health system in fostering integration of function. Workshop participants emphasized the importance of aligning goals and assessments and adopting a team science approach that includes clinicians and frontline staff in the planning, development, testing, and implementation of tools and initiatives. This article summarizes those discussions.     

Amino acid 489 is encoded by a mutational "hot spot" on the beta 3 integrin chain: the CA/TU human platelet alloantigen system

A new platelet alloantigen, termed CA, has recently been implicated in a case of neonatal alloimmune thrombocytopenia (NATP) in a Filipino family in Canada. Maternal anti-CA serum reacted with glycoprotein (GP) IIIa and maintained its reactivity after removal of high mannose carbohydrate residues from GPIIIa. The monoclonal antibody (MoAb) AP3 partially blocked binding of anti-CA to GPIIIa, suggesting that the CA polymorphism is proximal to the AP3 epitope. Platelet RNA polymerase chain reaction (PCR) was used to amplify the region of GPIIIa cDNA that encodes this region of the protein. DNA sequence analysis showed a G<==>A nucleotide substitution at base 1564 that results in an arginine (Arg) (CGG)<==>glutamine (Gln) (CAG) polymorphism in amino acid (AA) 489. Further analysis of PCR-amplified genomic DNA from 27 normal individuals showed that AA 489 is encoded by a mutational "hot spot" of the GPIIIa gene, as three different codons for the wild-type Arg489 of GPIIIa were also found. The codon usage for Arg489 was found to be: CGG (63%), CGA (37%), and CGC (< 1%). These frequency data were valuable in determining the relationship of the CA alloantigen to the serologically defined TU GPIIIa polymorphism that is present in low frequency in the Finish population. Analyses of PCR-amplified genomic DNA showed the CA and TU alloantigens to be identical at the molecular level. Definition of these new molecular variants of the beta 3 integrin chain should prove valuable in the diagnosis of NATP in these two geographically disparate populations, and it may also provide useful genetic markers for examining other pathologic variations of the GPIIb-IIIa complex.     

Identification of the intact insulin receptor using a sequence-specific antibody directed against the C-terminus of the beta-subunit

An antibody was raised against a synthetic peptide corresponding to the carboxyl-terminal amino acids of the human insulin receptor (Anti-R beta C). Immunoprecipitation of the human insulin receptor and immunoblotting to the beta-subunit by Anti-R beta C could be inhibited by competition with the corresponding peptide. However, even at saturating concentrations, anti-R beta C could not completely immunoprecipitate or immunodeplete insulin receptors compared to a human autoantibody (anti-R B2). Using receptor labeled directly by 125I, evidence of multiple forms of the beta-subunit was found. When the receptor could be immunoprecipitated by anti-R beta C, the beta-subunit migrated with an apparent mol wt (MW) of 96,000 (at or above the phosphorylase b MW marker). However, in preparations where anti-R beta C was not able to immunoprecipitate the insulin receptor, the beta-subunit migrated at a significantly lower MW of 91,000 (below phosphorylase b), as detected by immunoprecipitation with Anti-R B2. Intermediate forms could also be detected. Phosphorylation of partially purified insulin receptor did not affect is ability to be immunoprecipitated by anti-R beta C, although insulin-stimulated phosphorylation increased the apparent MW of the beta-subunit. However, insulin receptor that was phosphorylated in solubilized extracts of whole cells had a beta-subunit that migrated at lower MW and was not immunoprecipitated by anti-R beta C. One possible explanation for this is that the beta-subunit may be degraded during preparation. When the MW of insulin receptor that has been purified to homogeneity from human placenta is compared to our data, it is clear that many of these insulin receptor preparations contain lower MW beta-subunits. These results must be taken into account when the sites of phosphorylation and kinase activity of purified insulin receptor preparations are studied.     

Safety of 2 recombinant human immunodeficiency virus type 1 (HIV-1) envelope vaccines in neonates born to HIV-1-infected women.

To determine the safety of 2 candidate vaccines against human immunodeficiency virus type 1 (HIV-1), a randomized, placebo-controlled, multicenter trial compared low, medium, and high doses of the vaccines or an adjuvant among infants born to HIV-infected women. No local or systemic reactions of grade 2 or greater were reported 48 h after the subjects underwent immunization. Grade 3 or 4 chemistry toxicities occurred in 5 (3%) and grade 3 or 4 hematologic toxicities in 17 (11%) of 154 vaccinated subjects (not significantly different from 29 adjuvant recipients). CD4(+) cell percentages of < or = 20% occurred at least once in 9 vaccinated subjects and 1 control subject. Sustained CD4(+) cell percentages of < or = 20% occurred in 4 HIV-infected children. Fourteen infants (8%) were confirmed to be HIV-infected; median CD4(+) cell counts among these children were 2074, 1674, 1584, and 821 cells/mm(3) at birth and weeks 24, 52, and 104, respectively. Thus, both vaccines were safe and well tolerated in neonates, and there was no evidence of accelerated immunologic decline in HIV-infected infants.     

Biotherapeutic agents in the treatment of infectious diarrhea

Biotherapeutic agents offer unique advantages over traditional treatments for infectious diarrhea, and several have been shown to be effective (Table 4). These therapeutic microbial agents are most effective in types of infectious diseases that are associated with a disruption of the normal intestinal microecology (e.g., AAD, C. difficile disease). The impact of biotherapeutic agents on rotaviral diarrhea is of special clinical importance because this is the most common cause of pediatric diarrhea, and there is no defined treatment. Strong efforts need to be made to limit antibiotic exposure in children. Biotherapeutic agents offer a safe and effective nonantibiotic method of treating this important pathogen, especially after the withdrawal of a rotaviral vaccine from the market by the FDA. However, for many biotherapeutic agents, well-done, placebo-controlled trials still are lacking, and not all types of infectious diarrhea respond to these agents. Continued research in this innovative therapeutic area is warranted.     

Purification and characterization of factor VII 304-Gln: a variant molecule with reduced activity isolated from a clinically unaffected male

Factor VII (FVII) is the plasma serine protease zymogen which, on binding to its cellular receptor tissue factor (TF), initiates blood coagulation. A 47-year-old man with no clinical bleeding tendency was found to have undetectable plasma FVII activity when tested in a one- stage assay using rabbit brain TF, but 0.3 U/mL using recombinant human TF and 1.04 U/mL FVII antigen. Variant FVII purified from his plasma showed an identical migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to wild-type zymogen. By enzyme kinetic analysis the Km of the variant using FX as a substrate was 12-fold higher than that of normal FVII. Also, the variant FVII was unable to compete with wild-type FVII for limited rabbit TF binding sites. A ligand blot procedure was used to directly demonstrate reduced binding of recombinant human TF to the variant FVII compared with normal FVII. Genetic analysis of leukocyte DNA showed a G to A mutation in the propositus' gene at codon 304 that results in the substitution of a glutamine for an arginine residue in the catalytic domain of the protease. We conclude that this region of the FVII molecule is important for its function.     

Heterogeneity of T-cell receptor alpha-chain complementarity-determining region 3 in myelin basic protein-specific T cells increases with severity of multiple sclerosis.

The pathogenesis of multiple sclerosis (MS) is thought to involve a T-cell-mediated autoimmune process. Experimental allergic encephalomyelitis (EAE), an animal model resembling MS, can be induced by immunization with myelin antigens such as myelin basic protein. The T-cell antigen receptor (TCR) usage in EAE is highly restricted in some strains of animals and experimental treatments targeting the TCR have been successful in EAE. Examination of the TCR beta-chain variable-region (V beta) usage of MBP-specific T-cell lines in MS patients has produced conflicting results. Our previous studies of TCR alpha-chain variable-region usage in monozygotic twins demonstrated a general skewing of the TCR repertoire in individuals with MS. This skewing became apparent only after stimulation with antigens; in peripheral blood lymphocyte preparations from individuals with MS V alpha 8-bearing T cells were preferentially selected by stimulation with myelin basic protein. In the present study we examined complementarity-determining region 3 of those V alpha 8-positive TCRs. Marked sequence heterogeneity was found in all individuals with severe MS. In contrast, restricted areas of complementarity-determining region 3 were found in healthy control individuals and in individuals with a mild form of MS. Sequences from tetanus toxoid-specific V alpha 8-positive T cells generated from the same individuals were relatively homogeneous within individuals regardless of disease activity and were distinct from the sequences of complementarity-determining region 3 in myelin basic protein-stimulated lines. These findings suggest that disease severity may be associated with increased heterogeneity of myelin antigen-specific T cells and could reflect an impaired ability of the immune system to down-regulate these anti-self responses.     

Impact of a Single Session of Counseling on Partner Referral for Sexually Transmitted Disease Treatment, Harare, Zimbabwe

Counseling patients with sexually transmitted diseases (STD) to refer their partners to treatment is considered a means of preventing reinfection and controlling the spread of STD and is standard practice throughout the world. We conducted a randomized controlled trial to assess the impact of an enhanced counseling session on partner referral in Harare, Zimbabwe. The intervention consisted of an individualized confidential session with a trained counselor; standard care (control) relied on the treating clinician to discuss partner referral. A consecutive sample of 272 patients (135 men, 137 women) was randomly assigned to the intervention or control group; 137 (50%) completed follow- up. By intent-to-treat analysis, persons in the intervention arm were more likely to report notifying any partner compared to controls (92% vs. 67%, adjusted odds ratio 4.1, 95% confidence interval 1.3–13.2, p < .001). Across both study arms, women and married persons were more likely to notify partners, particularly spouses. Few persons notified casual partners. Qualitative data at follow-up identified diverse motivating factors and barriers to partner referral. A short, low-cost counseling session may increase the number of spouses referred to STD treatment in resource-poor, high-morbidity areas of sub-Saharan Africa. New methods of treating or notifying casual partners need to be evaluated.