Chlamydia trachomatis Infection of Endocervical Epithelial Cells Enhances Early HIV Transmission Events

Chlamydia trachomatis causes a predominantly asymptomatic, but generally inflammatory, genital infection that is associated with an increased risk for HIV acquisition. Endocervical epithelial cells provide the major niche for this obligate intracellular bacterium in women, and the endocervix is also a tissue in which HIV transmission can occur. The mechanism by which CT infection enhances HIV susceptibility at this site, however, is not well understood. Utilizing the A2EN immortalized endocervical epithelial cell line grown on cell culture inserts, we evaluated the direct role that CT-infected epithelial cells play in facilitating HIV transmission events. We determined that CT infection significantly enhanced the apical-to-basolateral migration of cell-associated, but not cell-free, HIV_BaL, a CCR5-tropic strain of virus, across the endocervical epithelial barrier. We also established that basolateral supernatants from CT-infected A2EN cells significantly enhanced HIV replication in peripheral mononuclear cells and a CCR5+ T cell line. These results suggest that CT infection of endocervical epithelial cells could facilitate both HIV crossing the mucosal barrier and subsequent infection or replication in underlying target cells. Our studies provide a mechanism by which this common STI could potentially promote the establishment of founder virus populations and the maintenance of local HIV reservoirs in the endocervix. Development of an HIV/STI co-infection model also provides a tool to further explore the role of other sexually transmitted infections in enhancing HIV acquisition.     

Staff Nurses’ Perceptions and Experiences about Structural Empowerment: A Qualitative Phenomenological Study

The aim of the study reported in this article was to investigate staff nurses’ perceptions and experiences about structural empowerment and perceptions regarding the extent to which structural empowerment supports safe quality patient care. To address the complex needs of patients, staff nurse involvement in clinical and organizational decision-making processes within interdisciplinary care settings is crucial. A qualitative study was conducted using individual semi-structured interviews of 11 staff nurses assigned to medical or surgical units in a 600-bed university hospital in Belgium. During the study period, the hospital was going through an organizational transformation process to move from a classic hierarchical and departmental organizational structure to one that was flat and interdisciplinary. Staff nurses reported experiencing structural empowerment and they were willing to be involved in decision-making processes primarily about patient care within the context of their practice unit. However, participants were not always fully aware of the challenges and the effect of empowerment on their daily practice, the quality of care and patient safety. Ongoing hospital change initiatives supported staff nurses’ involvement in decision-making processes for certain matters but for some decisions, a classic hierarchical and departmental process still remained. Nurses perceived relatively high work demands and at times viewed empowerment as presenting additional. Staff nurses recognized the opportunities structural empowerment provided within their daily practice. Nurse managers and unit climate were seen as crucial for success while lack of time and perceived work demands were viewed as barriers to empowerment.     

Specific binding of atrial natriuretic factor to renal glomeruli in Doca- and Doca-salt-treated rats correlation with atrial and plasma levels

Since volume expansion and high blood pressure (BP) are known stimuli of atrial natriuretic factor (ANF) release, and since this peptide may be involved in mineralocorticoid escape, we investigated the effects of chronic deoxycorticosterone (DOCA) and DOCA-NaC1 treatment on renal glomerular ANF receptor density and affinity in relation to atrial and plasma ANF levels. An increase in plasma immunoreactive ANF (IR-ANF) was observed both after two and four weeks of treatment. IR-ANF concentrations were elevated in the left atrium only in four-week DOCA treated rats. Administration of the mineralocorticoid alone resulted in a decreased density of glomerular ANF receptors in both time periods investigated. DOCA-NaC1-treated animals presented an increased receptor density during the pre-hypertensive stage (2 weeks) and a reduced density in the later hypertensive period (4 weeks). Receptor affinity in both groups was identical to that in the controls after 2 weeks and was augmented after 4 weeks of treatment. Our data suggest that the down-regulation of renal glomerular ANF receptors during chronic DOCA-NaC1 administration may play a role in the maintenance of high BP in this model of volume-expanded hypertension.     

The molecular mechanism of the bicarbonate effect at the plastoquinone reductase site of photosynthesis

It has been known for some time that bicarbonate reverses the inhibition, by formate under HCO₃ ^--depletion conditions, of electron transport in thylakoid membranes. It has been shown that the major effect is on the electron acceptor side of photosystem II, at the site of plastoquinone reduction. After presenting a historical introduction, and a minireview of the bicarbonate effect, we present a hypothesis on how HCO₃ ^- functions in vivo as (a) a proton donor to the plastoquinone reductase site in the D1-D2 protein; and (b) a ligand to Fe²⁺ in the Q_A-Fe-Q_B complex that keeps the D1-D2 proteins in their proper functional conformation. They key points of the hypothesis are: (1) HCO₃ ^- forms a salt bridge between Fe²⁺ and the D2 protein. The carboxyl group of HCO₃ ^- is a bidentate ligand to Fe²⁺, while the hydroxyl group H-bonds to a protein residue. (2) A second HCO₃ ^- is involved in protonating a histidine near the Q_B site to stabilize the negative charge on Q_B. HCO₃ ^- provides a rapidly available source of H⁺ for this purpose. (3) After donation of a H⁺, CO₃ ^2- is replaced by another HCO₃ ^-. The high pKa of CO₃ ^2- ensures rapid reprotonation from the bulk phase. (4) An intramembrane pool of HCO₃ ^- is in equilibrium with a large number of low affinity sites. This pool is a H⁺ buffering domain functionally connecting the external bulk phase with the quinones. The low affinity sites buffer the intrathylakoid [HCO₃ ^-] against fluctuations in the intracellular CO₂. (5) Low pH and high ionic strength are suggested to disrupt the HCO₃ ^- salt bridge between Fe²⁺ and D₂. The resulting conformational change exposes the intramembrane HCO₃ ^- pool and low affinity sites to the bulk phase.Two contrasting hypotheses for the action of formate are: (a) it functions to remove bicarbonate, and the low electron transport left in such samples is due to the left-over (or endogenous) bicarbonate in the system; or (b) bicarbonate is less of an inhibitor and so appears to relieve the inhibition by formate. Hypothesis (a) implies that HCO₃ ^- is an essential requirement for electron transport through the plastoquinones (bound plastoquinones Q_A and Q_B and the plastoquinone pool) of photosystem II. Hypothesis (b) implies that HCO₃ ^- does not play any significant role in vivo. Our conclusion is that hypothesis (a) is correct and HCO₃ ^- is an essential requirement for electron transport on the electron acceptor side of PS II. This is based on several observations: (i) since HCO₃ ^-, not CO₂, is the active species involved (Blubaugh and Govindjee 1986), the calculated concentration of this species (220 M at pH 8, pH of the stroma) is much higher than the calculated dissociation constant (Kd) of 35–60 M; thus, the likelihood of bound HCO₃ ^- in ambient air is high; (ii) studies on HCO₃ ^- effect in thylakoid samples with different chlorophyll concentrations suggest that the left-over (or endogenous) electron flow in bicarbonate-depleted chloroplasts is due to left-over (or endogenous) HCO₃ ^- remaining bound to the system (Blubaugh 1987).Abbreviations DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (common name: diuron) - PSII photosystem II - Q_A first plastoquinone electron acceptor of PSII - Q_B second plastoquinone acceptor of PS II     

A two-site immunoradiometric assay of proatrial natriuretic factor. Application to tissue extracts

A "two-site" immunoradiometric assay (IRMA) was developed to specifically measure ANF (1-126), the precursor of ANF. This assay is based on the simultaneous use of antibodies against two different antigenic determinants: murine monoclonal antibody (2H2), which recognizes positions 101 through 103 of ANF, is linked to Immunobeads and employed to extract any ANF C-terminal; a second antibody, which is directed against positions 11 through 37, is radioiodinated and allows binding to any C-terminal-2H2-Immunobead material which bears the N-terminal antigenic site. A curvilinear relationship was obtained between radioactivity and the amount of proANF (1.5 to 400 fmol) added. Optimisation of IRMA was determined by the amount of 2H2-Immunobeads and labeled antibody used, incubation time as well as possible interference by both ANF (99-126) and ANF (1-98). Tissue extracts were used to validate the assay. proANF was detected in decreasing amounts in heart atria, heart ventricles, lungs, kidneys and adrenal glands. Its presence was further confirmed by reverse-phase HPLC followed by radioimmunoassay. IRMA is a simple and rapid method for the direct measurement of proANF in tissue extracts and chromatographic fractions. The presence of proANF in tissues strongly suggests local synthesis.     

Comparative biological activities of ANF (Arg 101-Tyr 126) and the synthetic form of circulating ANF (Ser 99-Tyr 126)

The biological activities of ANF (Arg 101-Tyr 126) and of the circulating form, ANF (Ser 99-Tyr 126), were compared in the following assays: precontracted rabbit aortic strip and chick rectum, rat natriuresis, inhibition of aldosterone secretion and receptor affinity in bovine and rat adrenal zona glomerulosa cells, and receptor affinity in rabbit aorta and rat mesenteric artery cells. The results demonstrate that both peptides share the same biological activities. It is concluded that the addition of two amino acids to the N-terminal of ANF (Arg 101-Tyr 126) does not modify its biological characteristics, validating thus previous research employing this peptide.     

Partial characterization and solubilization of receptors for atrial natriuretic factor in rat glomeruli

Specific receptors for atrial natriuretic factor (ANF) have been identified and solubilized in glomeruli from rat kidney. Radioiodinated synthetic ANF (Arg 101-Tyr 126) bound to a single class of high affinity (Kd 27 +/- 24 pM) sites with a density of 390 +/- 230 fmole/mg protein. The binding was time- and temperature-dependent, saturable and reversible. The ANF-receptor complex was not affected by angiotensin II, ACTH or vasopressin. Solubilization with 10 mM 3-[(3-cholamidopropyl)-dimethylammonio]- 1-propane sulfonate (CHAPS) slightly increased the affinity for ANF (Kd 5.0 +/- 3.3 pM) without affecting the density (250 +/- 110 fmole/mg protein). Similar results were found with 1% Triton X-100. ANF-related peptides interact generally in the same way with non-solubilized and solubilized receptors, indicating a fully preserved specificity of the receptors.     

Identification of a biologically active circulating form of rat atrial natriuretic factor

An atrial natriuretic peptide has been isolated from plasma of morphine treated rats by means of glass beads extraction, immunoaffinity chromatography, and reverse phase HPLC. 1.3 micrograms of immunoreactive material was obtained. The biological activity of this material was found comparable to that of ANF (Arg 101 - Tyr 126) on the inhibition of basal aldosterone secretion by rat adrenal zona glomerulosa cells and the displacement curve of iodinated ANF from ANF receptors in a mesenteric artery preparation. Gas phase amino acid sequencing indicated that it is related to ANF (Ser 99 - Tyr 126). These results suggest that the maturation of ANF may require a tryptic-like cleavage after a single Arg residue.     

Characterization of specific receptors for atrial natriuretic factor in bovine adrenal zona glomerulosa

We have recently shown that synthetic rat atrial natriuretic factor (ANF) directly inhibits mineralocorticoid and glucocorticoid secretion in cultured bovine adrenal cells with a potency of 100 pM. [125I]iodo-ANF was used in the present study to characterize potential receptor sites in bovine zona glomerulosa membranes. ANF binds to a class of high affinity binding sites with a pK of 10.2 and a density of 1.3 pmol/mg protein. Detailed competition curves with ANF document a class of high affinity sites with a pK of 10.2 and also a second class of lower affinity sites with a pK of 8.5. Nonspecific binding amounts to less than 10% of [125I]iodo-ANF binding at concentrations less than 100 pM. High affinity binding of [125I]iodo-ANF is reversible with a half-time of association of 15 minutes at 25 pM and a half-time of dissociation of 140 minutes. Monovalent cations Na, Li and K equipotently enhance [125I]iodo-ANF specific binding. Divalent cations Mg, Ca and Mn also increase [125I]iodo-ANF specific binding, with Mn being the most active cation. No effect of guanine nucleotide could be detected on ANF binding. The binding of [125I]iodo-ANF is very specific and is not inhibited by 1 microM angiotensin II, ACTH, VIP, somatostatin, Leu-enkephalin, dynorphin or by the N-terminal of POMC. The N-terminal fragment ANF-(1-16) is also completely inactive. Reduction of the disulfide bridge of ANF inactivates the peptide. This enabled the development of a highly specific radio-receptor assay for ANF with a minimum detectable dose of 2 femtomoles. The results document the specific receptor involved in the potent inhibitory effect of ANF on adrenal steroidogenesis and indicate that bovine adrenal zonal glomerulosa provide a highly sensitive system for studying the recently discovered atrial natriuretic factor.     

The blood pressure decrease-induced sympathetic discharge following atrial natriuretic factor administration may offset its natriuretic action

Conscious SHR and WKY rats were infused during 7 days with ANF (Arg 101-Tyr 126), 100 ng/hr/rat, by means of miniosmotic pumps and their basal blood pressure (BP), changes in sodium excretion and urinary catecholamines compared with those at the last day of the infusion. The SHR initial BP of 181 +/- 3 mmHg gradually declined to 137 +/- 5 mmHg. No significant change in blood pressure was observed in the ANF-infused WKY group. However, WKY rats exhibited an increased sodium excretion and urinary dopamine/norepinephrine ratio when compared to sham-infused rats. No such differences were observed in SHR. It is suggested that an ANF-induced withdrawal of the renal sympathetic tone permits the manifestation of its natriuretic action in WKY rats. When, however, a BP decrease predominates, as in SHR, this decrease results in a reflex sympathetic discharge with a renal sympathetic activity over-riding the ANF induced natriuresis seen in WKY rats. Secondary sympathetic responses to the ANF-induced BP decrease have to be thus taken into account when a dissociation between the hypotensive and natriuretic action of ANF is observed in vivo.