Hydrophilic peptides derived from the transframe region of Gag-Pol inhibit the HIV-1 protease

The HIV-1 transframe region (TFR) is between the structural and functional domains of the Gag-Pol polyprotein, flanked by the nucleocapsid and the protease domains at its N and C termini, respectively. Transframe octapeptide (TFP) Phe-Leu-Arg-Glu-Asp-Leu-Ala-Phe, the N terminus of TFR, and its analogues are competitive inhibitors of the action of the mature HIV-1 protease. The smallest, most potent analogues are tripeptides: Glu-Asp-Leu and Glu-Asp-Phe with Ki values of approximately 50 and approximately 20 microM, respectively. Substitution of the acidic amino acids in the TFP by neutral amino acids and d or retro-d configurations of Glu-Asp-Leu results in an >40-fold increase in Ki. Protease inhibition by Glu-Asp-Leu is dependent on a protonated form of a group with a pKa of 3.8; unlike other inhibitors of HIV-1 protease which are highly hydrophobic, Glu-Asp-Leu is extremely soluble in water, and its binding affinity decreases with increasing NaCl concentration. However, Glu-Asp-Leu is a poor inhibitor (Ki approximately 7.5 mM) of the mammalian aspartic acid protease pepsin. X-ray crystallographic studies at pH 4.2 show that the interactions of Glu at P2 and Leu at P1 of Glu-Asp-Leu with residues of the active site of HIV-1 protease are similar to those of other product-enzyme complexes. It was not feasible to understand the interaction of intact TFP with HIV-1 protease under conditions of crystal growth due to its hydrolysis giving rise to two products. The sequence-specific, selective inhibition of the HIV-1 protease by the viral TFP suggests a role for TFP in regulating protease function during HIV-1 replication.     

Plasma membrane cholesterol is a key molecule in shear stress-dependent activation of extracellular signal-regulated kinase

Shear stress, the dragging force generated by fluid flow, differentially activates extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) in bovine aortic endothelial cells (BAEC) (Jo, H., Sipos, K., Go, Y. M., Law, R., Rong, J., and McDonald, J. M. (1997) J. Biol. Chem. 272, 1395-1401). Here, we examine whether cholesterol-enriched compartments in the plasma membrane are responsible for such differential regulation. Pretreatment of BAEC with a cholesterol-binding antibiotic, filipin, did not inhibit shear-dependent activation of JNK. In contrast, filipin and other membrane-permeable cholesterol-binding agents (digitonin and nystatin), but not the lipid-binding agent xylazine, inhibited shear-dependent activation of ERK. The effect of cholesterol-binding drugs did not appear to be due to membrane permeabilization, since treatment of BAEC with a detergent, Triton X-100 which also permeabilizes membranes, did not inhibit shear-dependent activation of ERK. Furthermore, shear-dependent activation of ERK, but not JNK, was inhibited by cyclodextrin, a membrane-impermeable cholesterol-binding agent, which removes cell-surface cholesterol. Moreover, the effects of cyclodextrin were prevented by adding cholesterol during the incubation. These results indicate that cholesterol or cholesterol-sensitive compartments in the plasma membrane play a selective and essential role in activation of ERK, but not JNK, by shear stress. Although exposure to shear stress (1 h) increased the number of caveolae by 3-fold, treatment with filipin had no effect in either control or shear-exposed cells suggesting that caveolae density per se is not a crucial determinant in shear-dependent ERK activation. In summary, the current study suggests that cholesterol-sensitive microdomains in the plasma membrane, such as caveolae-like domains, play a critical role in differential activation of ERK and JNK by shear stress.     

Effect of pulmonary artery stenoses on the cardiopulmonary response to exercise following repair of tetralogy of Fallot

Data from exercise tests, echocardiograms, and lung perfusion scans were analyzed to determine whether the excessive minute ventilation (VE) often encountered among patients with tetralogy of Fallot is due to ventilation-perfusion mismatch secondary to branch pulmonary artery stenoses. Patients with branch PA stenoses had lower peak oxygen consumptions and higher VE during exercise than did patients without stenoses, and a strong correlation existed between the degree of pulmonary blood flow maldistribution on lung perfusion scan and the amount of excessive VE during exercise.     

Indocyanine-green videoangiography of drusen as a possible predictive indicator of exudative maculopathy

OBJECTIVE: Recent studies have shown that indocyanine-green videoangiography (ICG-V) is useful to image occult choroidal neovascularization. The authors studied the ICG-V findings in fellow drusen eyes of patients with unilateral exudative age-related macular degeneration (AMD). The authors also studied the occurrence of exudative changes to determine whether ICG-V is useful in predicting future exudative changes in these eyes with only drusen. DESIGN: Cohort study. PARTICIPANTS: The authors studied 432 consecutive patients diagnosed with unilateral exudative AMD in whom the fellow eye had only drusen by clinical fundus examination and fluorescein angiography. All of these eyes had ICG-V performed. Follow-up data were obtained in all eyes with abnormal indocyanine-green (ICG) angiograms and randomly sampled ICG angiograms of normal eyes. MAIN OUTCOME MEASURES: The initial ICG findings were classified as showing normal or abnormal hyperfluorescence. Abnormal hyperfluorescence eyes were subdivided into focal spots (focal areas of hyperfluorescence < 1 disc area in size) and plaques (areas of hyperfluorescence > 1 disc area). The development of exudative changes in eyes with normal and abnormal hyperfluorescence was compared. RESULTS: Of the 432 fellow eyes, 386 (89%) eyes with drusen had a normal ICG-V study, whereas 46 (10 focal spots and 36 plaques) (11%) eyes had an abnormal ICG-V. Exudative changes occurred in 6 (10%) of 58 normal ICG eyes and 9 (24%) of 38 eyes with abnormal ICG findings during a mean follow-up period of 21.7 months. The difference between drusen eyes with normal ICG angiograms and those with plaques on ICG-V regarding future exudative changes (10% vs. 27%, respectively) was statistically significant (P = 0.038). CONCLUSIONS: Abnormal ICG findings were found in 11% of eyes with clinically and fluorescein angiographically nonsuspicious drusen. The subgroup of patients with plaques on ICG-V had a higher chance of having exudative changes develop. Indocyanine-green videoangiography may be a predictive indicator of future exudative changes in eyes with drusen. A much larger prospective study seems justified.     

Quantitation of adsorption and conjugation of plasma proteins by residual glutaraldehyde in fixed collagenous tissue with radioiodinated plasma proteins

Residual glutaraldehyde (GA) in collagenous cardiovascular tissue prostheses after multiple saline rinses remains in the prostheses and accounts for adsorption and conjugation of a variety of plasma proteins. This may account for later beneficial or adverse effects. Human serum albumin (SA), gamma globulin (GG), and fibrinogen (FB) were iodinated with 125I using the iodogen-transfer technique. Bovine pericardium (PC) was fixed with 0.5% GA for 24 hr and rinsed to remove excess GA. Fresh and GA-fixed PC (FRPC, GAPC: 1 x 1 cm2), in triplicate, were incubated with 0.5-1.0 microCi of tracers in human, porcine, or bovine blood (2 ml) for a period of 0.5, 1, 2, and 3 hr and washed (5x) with saline. Maximum adsorbed proteins per unit weight of collagen (pmol/mg of PC, mean +/- SD) at 3 hr on FRPC and GAPC were quantified with a gamma counter. Fixed PC absorbed significantly more plasma proteins from blood than fresh PC. These conjugated plasma proteins are tightly bound to fixed PC. The adsorbed and conjugated plasma proteins for GAPC and FRPC have the same sequence: SA > GG > FB vs SA > GG > FB. Protein conjugation may affect the remodeling of collagenous cardiovascular tissue prostheses post implantation.     

Aspects of oligonucleotide and peptide sequencing with MALDI and electrospray mass spectrometry

Biopolymer sequencing with mass spectrometry has become increasingly important and accessible with the development of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). Here we examine the use of sequential digestion for the rapid identification of proteolytic fragments, in turn highlighting the general utility of enzymatic MALDI ladder sequencing and ESI tandem mass spectrometry. Analyses were performed on oligonucleotides ranging in size from 2 to 50 residues, on peptides ranging in size from 7 to 44 residues and on viral coat proteins. MALDI ladder sequencing using exonuclease digestion generated a uniform distribution of ions and provided complete sequence information on the oligonucleotides 2-30 nucleic acid residues long. Only partial sequence information was obtained on the longer oligonucleotides. C-terminal peptide ladder sequencing typically provided information from 4 to 7 amino acids into the peptide. Sequential digestion, or endoprotease followed by exoprotease exposure, was also successfully applied to a trypsin digest of viral proteins. Analysis of ladder sequenced peptides by LCMS generated less information than in the MALDI-MS analysis and ESI-MS2 normally provided partial sequence information on both the small oligonucleotides and peptides. In general, MALDI ladder sequencing offered information on a broader mass range of biopolymers than ESI-MS2 and was relatively straightforward to interpret, especially for oligonucleotides.     

Regulation of an Escherichia coli/mammalian chimeric carbamoyl-phosphate synthetase

Carbamoyl-phosphate synthetase (CPSase) consists of a 120-kDa synthetase domain (CPS) that makes carbamoyl phosphate from ATP, bicarbonate, and ammonia usually produced by a separate glutaminase domain. CPS is composed of two subdomains, CPS.A and CPS.B. Although CPS.A and CPS.B have specialized functions in intact CPSase, the separately cloned subdomains can catalyze carbamoyl phosphate synthesis. This report describes the construction of a 58-kDa chimeric CPSase composed of Escherichia coli CPS.A catalytic subdomains and the mammalian regulatory subdomain. The catalytic parameters are similar to those of the E. coli enzyme, but the activity is regulated by the mammalian effectors and protein kinase A phosphorylation. The chimera has a single site that binds phosphoribosyl 5'-pyrophosphate (PRPP) with a dissociation constant of 25 microM. The dissociation constant for UTP of 0.23 mM was inferred from its effect on PRPP binding. Thus, the regulatory subdomain is an exchangeable ligand binding module that can control both CPS.A and CPS.B domains, and the pathway for allosteric signal transmission is identical in E. coli and mammalian CPSase. A deletion mutant that truncates the polypeptide within a postulated regulatory sequence is as active as the parent chimera but is insensitive to effectors. PRPP and UTP bind to the mutant, suggesting that the carboxyl half of the subdomain is essential for transmitting the allosteric signal but not for ligand binding.     

Age and ethnicity affect the risk and outcome of focal segmental glomerulosclerosis

In patients with proteinuria, African-American (AA) ethnicity is reported to be a risk factor for focal segmental glomerulosclereosis (FSGS) and its progression to end-stage renal disease (ESRD). We reviewed our single-center experience to determine the probability of FSGS and its progression to ESRD based on ethnicity and age at presentation in children with proteinuria with or without nephrotic syndrome. Proteinuria without systemic disease or acute glomerulonephritis was the presenting feature in 17% (236/1,403) of children in the renal patient database of Texas Children's Hospital, Baylor College of Medicine. Histopathological diagnoses were established in 107 of 236 patients (45%). FSGS was identified in 65 patients, accounting for 28% of all patients with proteinuria and 61% of patients who underwent renal biopsy. FSGS was more prevalent in AA (45%) than in non-AA patients (22%) (P=0.001), and AA patients with FSGS were older at presentation (12.7+/-4.4 years) than non-AA patients (5.6+/-4.6 years) (P<0.001). Among patients who underwent renal biopsy, increasing age at presentation increased the probability of having FSGS in AA but not non-AA patients (P=0.04). Five-year actuarial renal survival of FSGS was worse in AA (8%) than in non-AA patients (31%) (P=0.01). These data suggest an increased risk and worse outcome of FSGS in AA compared with non-AA children.     

Separation of the enantiomers of ibuprofen and its major phase I metabolites in urine using capillary electrophoresis

This report describes a simple, rapid, automated microassay for measuring in vitro changes of oxidative burst of phagocytes following challenge with metals for orthopedic devices. The production of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMNs) was measured using 2',7'-dichlorofluorescin-diacetate (DCFH-DA) as fluorescent probe. DCFH-DA enters the cells and is oxidized by ROS to fluorescent DCF. The DCF generated was directly proportional to ROS produced intracellularly: The fluorescence intensity was read and converted to an index of ROS production by cells. In our experimental system, granulocytes (PMNs) were isolated from normal human blood and seeded in microplates. To verify if metals could influence ROS production, chromium, cobalt, nickel, molybdenum, titanium, aluminum, and vanadium prepared as aqueous extracts in phosphate-buffered saline were tested onto PMNs using phorbolmyristate acetate (PMA) as positive control. Molybdenum, aluminum, and vanadium increased ROS generation by PMNs, while signals not different from unstimulated PMNs were recorded for chromium, cobalt, nickel, and titanium. The DCFH-DA microplate-based assay provides an in vitro tool for the detection of oxygen-reactive species generated by PMNs as a response to metals.     

Rapid multiplex analysis for the factor V Leiden and prothrombin G20210A mutations associated with hereditary thrombophilia

Thromboembolic episodes are common events and affect approximately one in 1,000 persons annually. Pulmonary embolism alone accounts for 50,000 to 100,000 deaths per year in the United States with > 50% of those being elderly persons. Resistance to activated protein C is the most common inherited disorder associated with hereditary thrombophilia. A missense mutation has been identified in the gene coding for coagulation factor V (codon 506) which renders this procoagulant factor resistant to inactivation by activated protein C resulting in an increased risk for venous thrombosis. Recently, a second polymorphism was identified in the prothrombin gene (factor II) which is also associated with increased risk for venous thrombosis. Because of the high prevalence of these two mutations in the general population as well as in specific patient populations, the ability readily to detect these two mutations must be feasible. In this study, we evaluated 303 patients for the prothrombin mutatin (G20210A) which were previously tested for the factor V mutation using established polymerase chain reaction-mediated restriction fragment length polymorphism assays. In these patients, 30 (9.9%) were found to be heterozygous for the factor V Leiden mutation with no homozygous mutants identified. Twenty individuals (6.6%) were heterozygous for the prothrombin G20210A mutation, and we identified two individuals (0.66%) who were homozygous for the 20210A allele. Of the total 303 individuals screened, two were double heterozygotes for both the factor V Leiden and the prothrombin gene mutations. We also describe a multiplex polymerase chain reaction-mediated restriction fragment length polymorphism assay for detecting both mutations in a single-tube double-enzyme digestion reaction making identification of these two mutations easily achievable.