Photoredox-Catalyzed Labeling of Hydroxyindoles with Chemoselectivity (PhotoCLIC) for Site-Specific Protein Bioconjugation.

We have developed a novel visible-light-catalyzed bioconjugation reaction, PhotoCLIC, that enables chemoselective attachment of diverse aromatic amine reagents onto a site-specifically installed 5-hydroxytryptophan residue (5HTP) on full-length proteins of varied complexity. The reaction uses catalytic amounts of methylene blue and blue/red light-emitting diodes (455/650 nm) for rapid site-specific protein bioconjugation. Characterization of the PhotoCLIC product reveals a unique structure formed likely through a singlet oxygen-dependent modification of 5HTP. PhotoCLIC has a wide substrate scope and its compatibility with strain-promoted azide-alkyne click reaction, enables site-specific dual-labeling of a target protein.

Implementing Lean Quality Improvement in Primary Care: Impact on Efficiency in Performing Common Clinical Tasks.

BACKGROUND: Many primary care practices have adopted Lean techniques to reduce the amount of time spent completing routine tasks. Few studies have evaluated both immediate and sustained impacts of Lean to improve this aspect of primary care work efficiency. OBJECTIVE: To examine 3-year impacts of Lean implementation on the amount of time taken for physicians to complete common clinical tasks. DESIGN: Non-randomized stepped wedge with segmented regression and interrupted time series analysis (January 2011-December 2016). PARTICIPANTS: A total of 317 physician-led teams in 46 primary care departments in a large ambulatory care delivery system. INTERVENTION: Lean redesign was initiated in one pilot site followed by system-wide spread across all primary care departments. Redesigns included standardization of exam room equipment and supplies, streamlining of call management processes, care team co-location, and team management of the electronic inbox. MEASURES: Time-stamped EHR tracking of physicians' completion time for 4 common tasks: (1) office visit documentation and closure of patient charts; (2) telephone call resolution; (3) prescription refill renewal; and (4) response to electronic patient messages. RESULTS: After Lean implementation, we found decreases in the amount of time to complete: office visit documentation (- 29.2% [95% CI: - 44.2, - 10.1]), telephone resolution (- 22.2% [95% CI: - 38.1, - 2.27]), and renewal of prescription refills (- 2.96% per month [95% CI: - 4.21, - 1.78]). These decreases were sustained over several years. Response time to electronic patient messages did not change significantly. CONCLUSIONS: Lean redesigns led to improvements in timely completion of 3 out of 4 common clinical tasks. Our findings support the use of Lean techniques to engage teams in routine aspects of patient care. More research is warranted to understand the mechanisms by which Lean promotes quality improvement and effectiveness of care team workflows.

Comparison of pharmacodynamic effects of ticagrelor vs prasugrel in type 2 diabetes mellitus patients with coronary heart disease.

WHAT IS KNOWN AND OBJECTIVE: Patients with type 2 diabetes mellitus (T2DM) are at higher risk of thrombotic complications. Studies have indicated that patients with T2DM have impaired clopidogrel-induced antiplatelet effect. Ticagrelor and prasugrel are two latest generation P2Y12 inhibitors with advantageous platelet inhibitory profiles. However, the pharmacodynamic differences between the two drugs in patients with T2DM remain poorly explored. METHODS: This study, involving 140 patients with T2DM following percutaneous coronary intervention (PCI), evaluated the efficacy of aspirin upon concomitant use of prasugrel (10 mg/d) or ticagrelor (90 mg/d). Platelet reactivity was assessed by value of ADP-induced light transmittance aggregometry (LTA) and vasodilator-stimulated phosphoprotein phosphorylation-platelet reactivity index (VASP-PRI) at baseline, 7 and 30 days after randomized P2Y12 inhibitor treatment. RESULTS: The study showed a decreased platelet reactivity after use of P2Y12 inhibitors (both P < .001). On the basis of comparison between regimens, apart from the prasugrel group having a significantly higher LTA value at the 30-day time point (P = .043), there existed no significant differences in platelet reactivity at separate time points (all P > .05). As for intragroup measurements, when compared with 7-day and 30-day time points, similar platelet reactivity was documented in the ticagrelor group (both P > .05), but LTA tests showed a significant increase with time (days 7-30) in the prasugrel group (P = .050). WHAT IS NEW AND CONCLUSION: Although ticagrelor and prasugrel have similar platelet inhibitory effects in patients with T2DM, if a P2Y12 inhibitor is necessitated in patients with T2DM, ticagrelor might exert a more stable antiplatelet effect with 30-day short-term treatment.

A Novel Rodent Orthotopic Forelimb Transplantation Model That Allows for Reliable Assessment of Functional Recovery Resulting From Nerve Regeneration.

Improved nerve regeneration and functional outcomes would greatly enhance the utility of vascularized composite allotransplantation (VCA) such as hand and upper extremity transplantation. However, research aimed at achieving this goal has been limited by the lack of a functional VCA animal model. We have developed a novel rat midhumeral forelimb transplant model that allows for the characterization of upper extremity functional recovery following transplantation. At the final end point of 12 weeks, we found that animals with forelimb transplantation including median, ulnar and radial nerve coaptation demonstrated significantly improved grip strength and forelimb function as compared to forelimb transplantation without nerve approximation (grip strength: 1.71N ± 0.57 vs. no appreciable recovery; IBB scale: 2.6 ± 0.7? vs. 0.8 ± 0.40; p = 0.0005), and similar recovery to nerve transection-and-repair only (grip strength: 1.71N ± 0.57 vs. 2.03 ± 0.42.6; IBB scale: 2.6 ± 0.7 vs. 2.8 ± 0.8; p = ns). Moreover, all forelimb transplant animals with nerve coaptation displayed robust axonal regeneration with myelination and reduced flexor muscle atrophy when compared to forelimb transplant animals without nerve coaptation. In conclusion, this is the first VCA small-animal model that allows for reliable and reproducible measurement of behavioral functional recovery in addition to histologic evaluation of nerve regeneration and graft reinnervation.

BRCA1 gene therapy reduces systemic inflammatory response and multiple organ failure and improves survival in experimental sepsis.

Sepsis-related complications and mortality remain a major clinical problem. Increased cell death and unresolved cellular repair have been implicated as key upstream mediators of sepsis-induced organ dysfunction and death. We hypothesised that gene therapy with BRCA1, a critical regulator of DNA damage repair and cell survival, would attenuate the sequelae of sepsis and peritonitis in mice subjected to caecal ligation and perforation (CLP) and thioglycollate stimulation. C57Bl/6J mice underwent sham or CLP surgery 3 days following treatment with either human BRCA1 adenovirus (AdBRCA1) or the adeno-CMV-null vector (Adnull). The 24-h post-CLP mortality was 2.8% vs 17.9% (P<0.001) and the median post-CLP survival was 50.5 vs 33 h (P<0.05) for AdBRCA1- vs Adnull-treated mice, respectively. AdBRCA1 therapy blunted CLP-associated cardiac, pulmonary, hepatic and renal dysfunction and also reduced CLP-elicited double strand breaks and apoptosis in the liver. BRCA1 gene therapy was associated with lower CLP-evoked cardiac and hepatic superoxide generation that in the liver was in part due to improved reactive oxygen species removal. CLP also elevated mesenteric arteriolar and serum intercellular adhesion molecule-1, both of which were partially abrogated with AdBRCA1 administration. Thioglycollate-challenged AdBRCA1-treated mice displayed reduced peritoneal neutrophil recruitment and dampened cytokine elaboration relative to their Adnull-treated counterparts. Taken together, we report a novel role of BRCA1 gene therapy in limiting systemic inflammation, multiple-organ failure and mortality in experimental sepsis.

Growth factors, signaling pathways, and the regulation of proliferation and differentiation in BC3H1 muscle cells. II. Two signaling pathways distinguished by pertussis toxin and a potential role for the ras oncogene.

In the preceding report (Kelvin, D.J., G. Simard, H.H. Tai, T.P. Yamaguchi, and J.A. Connolly. 1989. J. Cell Biol. 108:159-167) we demonstrated that pertussis toxin (PT) blocked proliferation and induced differentiation in BC3H1 muscle cells. In the present study, we have used PT to examine specific growth factor signaling pathways that may regulate these processes. Inhibition of [3H]thymidine by PT in 20% FBS was reversed in a dose-dependent fashion by purified fibroblast growth factor (FGF). In 0.5% FBS, the normally induced increase in creatine kinase (CK) activity was blocked by FGF in both the presence and absence of PT. Similar results were obtained with purified epidermal growth factor (EGF). We subsequently examined the effect of a family of growth factors linked to inositol lipid hydrolysis and found that thrombin, like FGF, would increase [3H]thymidine incorporation and block CK synthesis. However, PT blocked thymidine incorporation induced by thrombin, and blocked the inhibition of CK turn-on in 0.5% FBS by thrombin. The ras oncogene, a G protein homologue, has previously been shown to block muscle cell differentiation in C2 muscle cells (Olson, E.N., G. Spizz, and M.A. Tainsky. 1987. Mol. Cell. Biol. 7:2104-2111); we have characterized a BC3H1 cell line, BCT31, which we transfected with the val12 oncogenic Harvey ras gene. This cell line did not express CK in response to serum deprivation. Whereas [3H]thymidine incorporation was inhibited by 70-80% by increasing doses of PT in control cells, BCT31 cells were only inhibited by 15-20%. ADP ribosylation studies indicate this PT-insensitivity is not because of the lack of a PT substrate in this cell line. Furthermore, PT could not induce CK expression in BCT31 cells as it did in parental cells. We conclude that there are at least two distinct growth factor pathways that play a key role in regulating proliferation and differentiation in BC3H1 muscle cells, one of which is PT sensitive, and postulate that a G protein is involved in transducing signals from the thrombin receptor. We believe that ras functions in the transduction of growth factor signals in the nonPT-sensitive pathway or downstream from the PT substrate in the second pathway.

Contextual Conditions and Performance Improvement in Primary Care.

BACKGROUND: Although organizational context can affect the implementation of quality initiatives, we know less about the influence of contextual conditions on quality outcomes. We examined organizational features of primary care clinics that achieved greatest performance improvements after implementing Lean redesigns. METHODS: We used operational data and baseline (ie, pre-Lean implementation) surveys of 1333 physicians and staff in 43 primary care clinics located across a large ambulatory care system. Segmented regression with interrupted time series analysis was used to identify clinics with highest improvements in workflow efficiency, physician productivity, and patient satisfaction following Lean redesign. We conducted independent-samples t tests to identify contextual features of clinics that showed greatest improvements in performance outcomes. RESULTS: Clinics with highest increases in efficiency had most prior experience with quality improvement, compared with all other clinics. Efficiency gains were also found in clinics reporting highest levels of burnout and work stress prior to redesign. Highest improvements in physician productivity were associated with a history of change, staff participation, and leadership support for redesigns. Greatest improvements in patient satisfaction occurred in least stressful environments with highest levels of teamwork, staff engagement/efficacy, and leadership support. CONCLUSIONS: Our findings encourage careful evaluation of clinic characteristics and capacity to effectively implement redesigns. Such evaluations may help leaders select interventions most appropriate for certain clinics, while identifying others that may need extra support with implementing change.

Sustainment of Lean Redesigns for Primary Care Teams.

BACKGROUND: Quality improvements are notoriously followed by "backsliding" or relapse to the status quo. This mixed-methods study examined the sustainment of Lean workflow redesigns for primary care teams several years after being implemented in a large, ambulatory care delivery system. METHODS: We conducted qualitative interviews of 57 leaders and frontline providers, and fielded post-Lean implementation surveys to 1164 physicians and staff in 17 primary care clinics across the system. We analyzed interviews and conducted independent sample t tests to identify key factors that facilitated the sustainment of new workflows among primary care teams. All analyses were conducted after Lean redesigns were implemented and scaled across the system in 3 consecutive phases. RESULTS: Adherence to Lean redesigns was highest in the pilot clinic, despite having the longest postdesign measurement period. Members of the pilot clinic reported greatest participation in designing workflows, were most highly engaged in quality improvement efforts, and held most favorable beliefs about Lean changes. Adherence to redesigns was lowest among clinic members in the second phase of implementation; these members also reported highest levels of burnout. CONCLUSIONS: Staff participation in Lean redesign is a key to facilitating buy-in and adherence to changes. Change ownership and continued availability of time for improvement activities are also critical to the long-term success of Lean implementation in primary care.

Endogenous production of angiotensin II modulates rat proximal tubule transport.

There is evidence that angiotensin II is synthesized by the proximal tubule and secreted into the tubular lumen. This study examined the functional significance of endogenously produced angiotensin II on proximal tubule transport in male Sprague-Dawley rats. Addition of 10(-11), 10(-8), and 10(-6) M angiotensin II to the lumen of proximal convoluted tubules perfused in vivo had no effect on the rate of fluid reabsorption. The absence of an effect of exogenous luminal angiotensin II could be due to its endogenous production and luminal secretion. Luminal 10(-8) M Dup 753 (an angiotensin II receptor antagonist) resulted in a 35% decrease in proximal tubule fluid reabsorption when compared to control (Jv = 1.64 +/- 0.12 nl/mm.min vs. 2.55 +/- 0.32 nl/mm.min, P < 0.05). Similarly, luminal 10(-4) M enalaprilat, an angiotensin converting enzyme inhibitor, decreased fluid reabsorption by 40% (Jv = 1.53 +/- 0.23 nl/mm.min vs. 2.55 +/- 0.32 nl/mm.min, P < 0.05). When 10(-11) or 10(-8) M exogenous angiotensin II was added to enalaprilat (10(-4) M) in the luminal perfusate, fluid reabsorption returned to its baseline rate (Jv = 2.78 +/- 0.35 nl/mm.min). Thus, addition of exogenous angiotensin II stimulates proximal tubule transport when endogenous production is inhibited. These experiments show that endogenously produced angiotensin II modulates fluid transport in the proximal tubule independent of systemic angiotensin II.

Acute impairment of relaxation by low levels of testosterone in porcine coronary arteries.

OBJECTIVES: While there are many suggested reasons for the marked gender bias in cardiovascular events, much of the available data indicate that circulating estrogens are cardioprotective. The possibility that endogenous androgens may be detrimental to the cardiovascular system has received relatively less attention. We investigated the short-term modulatory effects of various concentrations of testosterone on vascular function in isolated porcine coronary artery rings. RESULTS: The higher concentrations (> 1 microM) of testosterone relaxed U46619-contracted coronary artery rings in an endothelium-independent manner. This direct effect was insensitive to the testosterone receptor antagonists, flutamide and cyproterone acetate. Short-term exposure (20 min) to low levels of testosterone (1-100 nM), which were ineffective on their own on vascular function, significantly diminished relaxation to bradykinin and calcium ionophore A23187 but not those produced by levcromakalim and sodium nitroprusside. The inhibitory effect observed with 1 nM testosterone was only partially reversed by flutamide and cyproterone acetate and unaltered in the presence of actinomycin D and cycloheximide. CONCLUSIONS: These results demonstrate that acute treatment with testosterone, at concentrations that have no effect on their own, reduces vasorelaxation. Furthermore, they suggest that this modulatory action may be in part independent of the classical testosterone receptor since it was not completely sensitive to the anti-androgens and was not inhibited by the transcriptional and translational inhibitors. These findings support the postulation that testosterone may have unfavorable influences on vascular function.

Predictive value of quantitative PCR-based viral burden analysis for eight human herpesviruses in pediatric solid organ transplant patients.

Human herpesviruses can cause significant morbidity and mortality in pediatric solid organ transplant recipients. It was hypothesized that viral burden quantification by polymerase chain reaction using an internal calibration standard could aid in distinguishing between viral disease and latency. Here we report the results of a 2-year prospective study of 27 pediatric solid organ (liver, kidney, or heart) transplant recipients in which multiple samples were analyzed for levels of all eight human herpesviruses by internal calibration standard-polymerase chain reaction. Herpes simplex viruses 1 and 2, varicella-zoster virus, and Kaposi's sarcoma-associated herpesvirus were not detected in any of these samples. Human herpesvirus types 6 and 7 were detected in half of the patients, but were present at low levels, similar to those found in reference populations. Epstein-Barr virus (EBV) and cytomegalovirus (CMV) were detected in 89% and 56% of the patients, respectively. Viral burden analysis suggested distinct patient populations for CMV, with a natural cutoff of 10,000 viral targets/ml blood strongly associated with disease. In some cases, a dramatic increase in CMV levels preceded clinical evidence of disease by several weeks. EBV viral burden was relatively high in the only patient presenting with an EBV syndrome. However, two other patients without evidence of EBV disease had single samples with high EBV burden. Rapid reduction in both EBV and CMV burden occurred with antiviral treatment. These data suggest that viral burden analysis using internal calibration standard-polymerase chain reaction for CMV, and possibly other herpesviruses, is an effective method for monitoring pediatric transplant patients for significant herpesvirus infection and response to therapy.

Recurrence of hemolytic uremic syndrome after renal transplantation in children: a report of the North American Pediatric Renal Transplant Cooperative Study.

BACKGROUND: Hemolytic uremic syndrome (HUS) is the cause of renal failure in 2-4% of children on dialysis. After renal transplantation, HUS can recur, but recurrence rate and risk factors are controversial. METHODS: We reviewed the recurrence of HUS within the North American Pediatric Renal Transplant Cooperative Study (NAPRTCS) registry and used a separate questionnaire to ascertain additional clinical information. RESULTS: Of 68 renal allografts, HUS recurred in 6 allografts (8.8%) occurring in five patients (8.2%). Four patients had atypical HUS, whereas one patient had classic HUS. HUS recurred after transplantation in 33 days or less in all but one allograft. Outcome was poor with five of six allografts lost, despite treatment with fresh-frozen plasma or plasmapheresis. Cyclosporine had no effect on outcome or HUS recurrence. CONCLUSIONS: The risk of HUS recurrence in the allograft is 8-9% and is heightened in atypical HUS. Treatment was not effective and graft outcome was poor. Cyclosporine does not affect HUS recurrence.

Effects of a centrally active benzoylpyrrolidine drug on AMPA receptor kinetics.

A newly developed benzoylpyrrolidine drug (BDP-20) that increases the size of fast, excitatory synaptic responses was examined for its effects on the kinetic properties of alpha-amino-3-hydroxy-5-methyl-4-isoxalepropionic acid (AMPA)-type glutamate receptors. When long pulses of glutamate were applied to excised hippocampal patches of the rat, the compound BDP-20 caused an approximately 15-fold reduction in the rate at which responses desensitized and a similar size increase in steady-state currents. In experiments using 1-ms glutamate pulses, BDP-20 prolonged response deactivation by a factor of about four and greatly reduced the depression in the second response when two consecutive glutamate pulses were given. Two types of equilibrium binding assays indicated that BDP-20 causes a measurable increase in the affinity of AMPA receptors; the EC50 values for this effect were similar to those obtained in excised patch studies. The actions of BDP-20 on physiology and ligand binding could be adequately reproduced in a receptor model by slowing the rate of desensitization and increasing the affinity of the sensitized states. The biochemical and physiological effects of this benzoylpyrrolidine compound were qualitatively different from those obtained with cyclothiazide, although both types of drug increased AMPA receptor-mediated synaptic responses. Moreover, interactions between the drugs were at most only partially competitive; AMPA receptors may thus have multiple modulatory sites with distinct drug preferences and different effects on receptor kinetics.

Differential effects of 17beta-estradiol and testosterone on the contractile responses of porcine coronary arteries.

1. We investigated the effects of short-term exposure to physiological levels of 17beta-estradiol and testosterone on vasocontractile responses in porcine coronary artery rings. 2. Concentration-response curves to endothelin-1, 5-hydroxytryptamine, the thromboxane analogue U46619 and KCl were constructed in endothelium-intact and endothelium-disrupted artery rings. 3. Thirty minutes exposure to 17beta-estradiol (1 and 30 nM) significantly attenuated vasoconstriction to endothelin-1, 5-hydroxytryptamine and U46619. Conversely, the same concentrations of testosterone significantly potentiated responses elicited by these contractile agents. These inhibitory effects of 17beta-estradiol and enhancing actions of testosterone on contractions were endothelium-independent. KCl-mediated contractions were unaffected by the presence of either sex hormones. 4. The oestrogen receptor antagonists, tamoxifen (10 microM) and ICI 182,780 (10 microM), were unable to reverse the inhibitory influence 1 nM 17beta-estradiol had on the agonist-mediated contractile responses. Similarly, the androgen receptor antagonists, flutamide (10 microM) and cyproterone acetate (10 microM), failed to affect the potentiating activities of 1 nM testosterone. The alteration in vasoconstrictive responses observed following acute exposure to either 1 nM 17beta-estradiol and 1 nM testosterone were apparent even in the presence of the protein synthesis inhibitor cycloheximide (10 microM) and the transcription inhibitor actinomycin D (10 microM). 6. In conclusion, we report a unique type of sex hormone action on the coronary vasculature. These events occur at low nanomolar concentrations of 17beta-estradiol and testosterone, are insensitive to conventional sex hormone receptor antagonists, are not blocked by de novo protein synthesis inhibitors and have rapid time-courses that are uncharacteristic of classical genomic activities.

Acute vision loss in children with autosomal recessive polycystic kidney disease.

Patients with autosomal recessive polycystic kidney disease (ARPKD) often present with renal insufficiency and hypertension. We present two children with ARPKD and end-stage renal disease who developed anterior ischemic optic neuropathy and vision loss. Anterior ischemic optic neuropathy occurs rarely in children and has never been reported in children with ARPKD or end-stage renal disease. Both of our patients were chronically hypotensive and anemic, which are known risk factors for ischemic optic neuropathy.

Regulation of proximal tubule transport by angiotensin II.

Angiotensin II maintains extracellular volume homeostasis, in part, by regulating proximal tubule transport. Physiological doses of angiotensin II stimulate volume and solute transport in the proximal tubule independent of changes in the glomerular filtration rate. Stimulation of bicarbonate transport primarily occurs via increasing activity of the sodium/hydrogen exchanger and the sodium/bicarbonate cotransporter. The effects of circulating angiotensin II are mediated by angiotensin II receptors on the basolateral membrane of the proximal tubule. Recently, the proximal tubule was found to synthesize and secrete angiotensin II into the lumen. The luminal membrane contains angiotensin II receptors and luminal angiotensin II levels are 100 to 200-fold higher than that found in plasma. Luminal angiotensin II receptor blockade or luminal inhibition of angiotensin II synthesis both significantly diminish proximal tubule transport, consistent with stimulation of proximal tubule transport by endogenously produced and luminally secreted angiotensin II. These data provide evidence for an autocrine/paracrine role for angiotensin II that functions independent of circulating angiotensin II.

Effect of luminal angiotensin II receptor antagonists on proximal tubule transport.

The proximal tubule can endogenously synthesize and secrete luminal angiotensin II at a concentration approximately 100- to 1000-fold higher than that in the systemic circulation. We have recently shown that this endogenously produced and luminally secreted angiotensin II regulates proximal tubule volume reabsorption, which is a reflection of sodium transport within this segment. In this study, we use in vivo microperfusion of angiotensin II receptor antagonists into the lumen of the proximal tubule to examine the role of the luminal AT1 and AT2 receptor in the regulation of volume reabsorption. Systemically administered (intravenous) AT1 and AT2 receptor antagonists, acting through basolateral angiotensin II receptors, have previously been shown to inhibit proximal tubule transport. Luminal perfusion of 10(-6) mol/L Dup 753 (AT1 antagonist) and 10(-6) mol/L PD 123319 (AT2 antagonist) decreased proximal tubule volume reabsorption from 2.94 +/- 0.18 to 1.65 +/- 0.18 and 1.64 +/- 0.19 nL/mm x min, respectively, P < .01. Luminal perfusion of 10(-4) mol/L CGP 42112A, another AT2 antagonist, similarly decreased volume reabsorption to 1.32 +/- 0.36 nL/nm x min, P < .01. The inhibition of transport with AT1 and AT2 antagonist was additive, as luminal perfusion of 10(-6) mol/L Dup 753 plus 10(-6) mol/L 123319 resulted in a decrease in volume reabsorption to 0.41 +/- 0.31 nL/mm x min, P < .001 v control, P < .05 v Dup 753, and P < .01 v PD 123319. These results show that endogenously produced angiotensin II regulates proximal tubule volume transport via both luminal AT1 and AT2 receptors.

Cyclothiazide decreases [3H]AMPA binding to rat brain membranes: evidence that AMPA receptor desensitization increases agonist affinity.

The effects of cyclothiazide, a drug which blocks AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor desensitization, were tested on binding of [3H]AMPA to rat brain membranes. Cyclothiazide reduced [3H]AMPA binding by lowering the apparent affinity of the AMPA receptor. The magnitude of the decrease was temperature dependent and greater for membrane-bound than for solubilized receptors. These data provide evidence that desensitization increases the affinity of the AMPA receptor for agonists and indicate that a significant percentage of AMPA receptors in conventional equilibrium binding assays are in a desensitized state.

Effects of heparin on the properties of solubilized and reconstituted rat brain AMPA receptors.

Heparin was found to bind to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors and to alter their functional properties. AMPA receptors solubilized in 0.4% Triton X-100 bound to a heparin-agarose column and were eluted by 0.4 M NaCl. Soluble heparin inhibited 10 nM [3H]AMPA binding to detergent-solubilized receptors by 75% (IC50 = 10 micrograms/ml), but had little effect on binding to membrane-associated receptors. The inhibition of [3H]AMPA binding to detergent-solubilized receptors was not observed when binding was measured in the presence of 0.4 M NaCl, and no effect of heparin was observed on binding of the AMPA receptor antagonist [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Scatchard analyses of [3H]AMPA binding to solubilized receptors revealed that the inhibition induced by heparin was caused by a decrease in the apparent affinity of a portion of the total binding sites. Studies on AMPA receptors reconstituted in artificial lipid bilayers indicated that 10 micrograms/ml heparin enhanced cooperativity between channels and prolonged the lifetime of the open channel, but did not affect the amplitude of single channel currents. Thus, heparin may be added to the list of compounds known to modulate AMPA receptor function. These data also raise the possibility that heparin-containing proteoglycans, which are known to be concentrated at synaptic junctions, might be able to bind AMPA receptors and influence their functional characteristics.

Current techniques in cell and molecular biology.

Recent advances in the field of molecular biology have revolutionized our understanding of the functioning of living organisms and facilitated the development of robust tools for both diagnosis and treatment of diseases. With particular reference to the field of critical care medicine, development of molecular biology techniques have aided in the following: (1) rapid and highly specific detection of pathogenic infectious agents (eg, Mycobacterium tuberculosis, Pneumocystis carinii, cytomegalovirus, Legionella); (2) development of assays for measurement of circulating cytokines such as tumor necrosis factor (TNF) and interleukin (IL)-1 that has helped our understanding of the pathogenesis of the sepsis syndrome; (3) administration of antibodies or soluble receptors to attempt to prevent untoward effects of cytokines such as TNF or IL-1; and (4) the administration of recombinant deoxyribonucleic acid (DNA) or proteins to patients in an attempt to alter the course of a disease such as antioxidant enzymes (superoxide dismutase). The rapidity of progress in this field has been staggering, which necessitates frequent updating of our knowledge for clinicians to put these molecular tools to their best use. This brief review attempts to explain the basic principles of commonly used techniques in molecular biology including recombinant DNA, polymerase chain reaction, DNA libraries, gene therapy, and protein biochemistry in a manner that is understandable to those without an in-depth knowledge of the field.