Subnanosecond motions of tryptophan residues in proteins

The dynamics of protein molecules in the subnanosecond and nanosecond time range were investigated by time-resolved fluorescence polarization spectroscopy. Synchrotron radiation from a storage ring was used as a pulsed light source to excite the single tryptophan residue in a series of proteins. The full width at half maximum of the detected light pulse was 0.65 nsec, making it feasible to measure emission anisotropy kinetics in the subnanosecond time range and thereby to resolve internal rotational motions. The proteins investigated exhibit different degrees of rotational freedom of their tryptophan residue, ranging from almost no mobility to nearly complete freedom in the subnanosecond time range. The tryptophan residue of Staphylococcus aureus nuclease B (20,000 daltons) has a single rotational correlation time (varphi) of 9.9 nsec at 20 degrees C, corresponding to a rotation of the whole protein molecule. By contrast, bovine basic A1 myelin protein (18,000 daltons) exhibits varphi of 0.09 and 1.26 nsec, showing that the tryptophan residue in this protein is highly flexible. The single tryptophan of human serum albumin (69,000 daltons) has almost no rotational freedom at 8 degrees C (varphi = 31.4 nsec), whereas at 43 degrees C it rotates rapidly (varphi(1) = 0.14 nsec) within a cone of semiangle 26 degrees in addition to rotating together with the whole protein (varphi(2) = 14 nsec). Of particular interest in the large angular range (semiangle, 34 degrees ) and fast rate (varphi(1) = 0.51 nsec) of the rotational motion of the tryptophan residue in Pseudomonas aeruginosa azurin (14,000 daltons). This residue is known to be located in the hydrophobic interior of the protein. The observed amplitudes and rates of these internal motions of tryptophan residues suggest that elementary steps in functionally significant conformational changes may take place in the subnanosecond time range.     

Antagonistic selection from predators and pathogens alters food-web structure

Selection can alter predator-prey interactions. However, whether and how complex food-webs respond to selection remain largely unknown. We show in the field that antagonistic selection from predators and pathogens on prey body-size can be a primary driver of food-web functioning. In Windermere, U.K., pike (Esox lucius, the predator) selected against small perch (Perca fluviatilis, the prey), while a perch-specific pathogen selected against large perch. The strongest selective force drove perch trait change and ultimately determined the structure of trophic interactions. Before 1976, the strength of pike-induced selection overrode the strength of pathogen-induced selection and drove a change to larger, faster growing perch. Predation-driven increase in the proportion of large, infection-vulnerable perch presumably favored the pathogen since a peak in the predation pressure in 1976 coincided with pathogen expansion and a massive perch kill. After 1976, the strength of pathogen-induced selection overrode the strength of predator-induced selection and drove a rapid change to smaller, slower growing perch. These changes made perch easier prey for pike and weaker competitors against juvenile pike, ultimately increasing juvenile pike survival and total pike numbers. Therefore, although predators and pathogens exploited the same prey in Windermere, they did not operate competitively but synergistically by driving rapid prey trait change in opposite directions. Our study empirically demonstrates that a consideration of the relative strengths and directions of multiple selective pressures is needed to fully understand community functioning in nature.     

The role of inheritance and environment in predisposition to vascular disease in people of African descent.

OBJECTIVES: This study sought to compare vascular reactivity and carotid intima media thickness (CIMT) between Afro-Caribbean people in the United Kingdom (UK) and the West Indies and Afro-Caribbean and Caucasian people in the UK. BACKGROUND: Attenuated vascular reactivity and increased CIMT in black patients is seen as evidence for predisposition to vascular disease, but no comparisons exist between Afro-Caribbean people in different settings, which can provide insight into non-inherited determinants of increased ethnic susceptibility. METHODS: A representative community sample of 81 healthy Afro-Caribbean people and 101 Caucasian people in the UK was compared with 197 matched Afro-Caribbean people in Jamaica. Small vessel reactivity was assessed by measuring the absolute change from baseline in the reflection index (RI) of the digital volume pulse during intravenous infusion of albuterol (5 microg/min, DeltaRI(ALB)) and glyceryl trinitrate (5 microg/min, DeltaRI(GTN)). The CIMT was measured ultrasonographically in the distal 1 cm of the common carotid artery. RESULTS: Mean DeltaRI(ALB) was 4.2 percentage points (95% confidence interval [CI], 2.3 to 6.1, p < 0.001) lower in UK Afro-Caribbean people compared with Jamaican Afro-Caribbean people and 2.6 percentage points (95% CI, 0.4 to 4.7, p = 0.02) lower compared with Caucasian people, after adjusting for vascular risk profile. Adjusted mean CIMT of UK Afro-Caribbean people was 0.13 mm (95% CI, 0.08 to 0.17, p < 0.001) greater compared with Jamaican Afro-Caribbean people and 0.05 mm (95% CI, 0.01 to 0.10, p = 0.02) greater compared with Caucasian people. CONCLUSIONS: Healthy UK Afro-Caribbean people have greater and Jamaican Afro-Caribbean people have less impairment of vascular reactivity and intima media thickness compared with UK Caucasian people, suggesting that potentially modifiable environmental interactions may contribute to excess vascular disease in Afro-Caribbean people.     

Site-directed mutagenesis of platelet glycoprotein Ib alpha demonstrating residues involved in the sulfation of tyrosines 276, 278, and 279

The interaction between platelet glycoprotein (GP) Ib alpha and von Willebrand factor (VWF) is essential for initiation of hemostasis. The sulfation of the 3 tyrosine residues 276, 278, and 279 in GPIb alpha is an important posttranslational modification that seems to promote the interaction with VWF. The environment where sulfation of tyrosines occurs has been proposed to contain highly acidic residues. This investigation has examined the highly acidic region from Asp249 to Asp287 in the mature GPIb alpha protein. Changes to most of the carboxylic acids in this region resulted in decreased reactivity to VWF. Only 3 mutants (Glu270Gln, Asp283Asn, Asp283Asn/Glu285Gln/Asp287Asn) resulted in the abolition of sulfation. Two novel mutations were also created. First, a deletion of the 7 amino acids from Tyr276 to Glu282 led to a loss of sulfation and totally abolished VWF binding in the presence of botrocetin. This confirms that it is these 3 tyrosines that undergo sulfation and that this region is crucial for botrocetin-mediated VWF binding. The second mutation involves changing the lysine residues at 253, 258, and 262 to alanine. This also led to distinct changes in VWF binding and abolition of sulfation.     

Increased p21(CIP1/WAF1) and B cell lymphoma leukemia-x(L) expression and reduced apoptosis in alveolar macrophages from smokers

Alveolar macrophages (AMs) are the predominant defense cells in the airway, and their numbers are increased in smokers and subjects with chronic obstructive pulmonary disease. This increase may result from increased recruitment, increased proliferation, or reduced cell death. Apoptosis regulates inflammatory cell survival, and p21(CIP1/WAF1) is an important inhibitory regulator of cycle progression after oxidative stress. We have investigated whether chronic smoke exposure influences the expression and localization of cell cycle and apoptotic proteins in AM and bronchial epithelial cells in vivo. The increased numbers of AMs seen in smokers were only partially due to enhanced proliferation. p21(CIP1/WAF1) protein expression was increased in AMs and biopsies isolated from smokers and was found predominantly within the cytoplasm. In addition, B cell lymphoma leukemia (Bcl)-x(L), an antiapoptotic regulator, was also highly expressed in macrophages from smokers compared with nonsmokers and subjects with asthma. Hydrogen peroxide, an oxidative stress, induced cytoplasmic expression of p21(CIP1/WAF1) and failed to induce apoptosis in an in vitro model. These results suggested that AM and bronchial epithelial cells from smokers, in contrast to those from normal subjects and subjects with asthma, have reduced cell death. Thus, oxidative stress induced by cigarette smoking may contribute to the chronicity of inflammation in the airway, through a reduction of apoptosis.     

An interplay of genetic and environmental factors in familial hepatitis and myasthenia gravis

A family is described in which there occurred two cases of the lupoid type of active chronic hepatitis with cirrhosis, one of chronic persistent hepatitis, and one of myasthenia gravis. The two cases of lupoid hepatitis were in the proposita, a schoolgirl aged 16 years, and her great-aunt aged 69 years whom she had never met. The case of myasthenia gravis was that of the father. The whole family, except the great-aunt, had been exposed to an epidemic of infectious hepatitis five years previously, and the girl and her brother had contracted this disease. The schoolgirl later developed active chronic hepatitis while her brother had chronic persistent hepatitis without immunological concomitants.APART FROM COINCIDENCE, SOME COMBINATION OF THREE PROCESSES WAS REQUIRED TO ACCOUNT FOR THE ILLNESSES IN THIS FAMILY: a genetic predisposition to chronic liver disease in particular, a genetic predisposition to autoimmune reactions in general, and a ;triggering' effect of infection with the hepatitis virus.     

Antibody to two forms of dihydrolipoamide acetyltransferase (PDC-E2) in primary biliary cirrhosis

ABSTRACT— Dihydrolipoamide acetyltransferase, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), is the major autoantigen in primary biliary cirrhosis. By immunoblotting with sera from patients with primary biliary cirrhosis, we observed a double band, of molecular weight 70 and 74 kD for PDC-E2, when a preparation of bovine heart mitochondria was not boiled prior to electrophoresis. This double band could also be detected using antisera raised in rats or rabbits against intact PDC or PDC-E2, but not in antisera raised against a synthetic decamer representing the lipoic acid binding sequence of PDC-E2; the latter reacted only with the 74 kD component. Antibody eluted from either the 70 or 74 kD component reacted with both 70 and 74 kD components. By ELISA, sera from patients with primary biliary cirrhosis reacted more strongly with a non-boiled than a boiled PDC-E2, whereas immune animal sera reacted equally with both preparations. Thus, according to whether preparations of PDC are boiled or not, two conformationally alternative forms of the PDC-E2 protein can be revealed by immunoblotting. The two forms in non-boiled preparations migrate at molecular weights corresponding to 70 and 74 kD.