Recent insights into biological regulation from cell-free protein-synthesizing systems

We review the important role that cell-free protein-synthesizing systems (CFPSS) have played in the history of modern biology, and highlight two recent applications that illustrate their continued utility for the exploration of otherwise intractable aspects of gene expression and its regulation. Viral capsid assembly recreated in CFPSS reveals a catalyzed biochemical pathway involving transient, energy-dependent action of host proteins and discrete assembly intermediates, rather than the classical notion of self-assembly that was expected for capsid formation. Study of prion protein biogenesis reveals a new conformation critical for disease pathogenesis and advances the paradigm of protein bioconformatics, by which cells may productively regulate the folding of various proteins. In each example, the CFPSS made it easier to analyze biochemical mechanism than is possible in other currently available whole cell systems, illustrating why this approach is likely to be a continuing source of insight into important features of biological regulation.     

Knee joint moments during stair climbing of patients with anterior cruciate ligament deficiency

OBJECTIVE: To establish the gait adaptations of patients with anterior cruciate ligament deficiency during stair ascent. DESIGN: Joint kinematics and kinetics during stair climbing were measured in both knees of normal subjects and unilateral anterior cruciate ligament deficient patients. BACKGROUND: As there is limited research pertaining to activities other than level walking, the purpose of the current study was to elucidate the gait of patients with anterior cruciate ligament deficiency during stair climbing to determine the effects of the deficiency on knee joint motion and moments as compared with normal knee function. METHODS: A motion analysis system was used to measure and calculate kinematic and kinetic data for six normal subjects and nine patients with unilateral anterior cruciate ligament deficiency during stair ascent on a specially constructed staircase. Left and right leg data were analysed to reveal between-limb differences for each subject. RESULTS: Patients with anterior cruciate ligament deficiency displayed a significant (P < 0.05) reduction of up to 50% in peak knee flexion moments in their involved knee. In the present study where a step height of 15.5 cm was used, peak flexion moments in all subjects' limbs occurred at knee flexion angles of about 40 degrees during single limb support. CONCLUSION: The current study showed that most patients with an anterior cruciate deficient knee adapted their gait during stair ascent. RELEVANCE: As stair climbing is a common activity, its effect on gait is relevant to better understand appropriate treatment and management strategies of patients with anterior cruciate ligament deficient knees.     

Localization of shapes: eye movements and perception compared

The localization of spatially extended objects is thought to be based on the computation of a default reference position, such as the center of gravity. This position can serve as the goal point for a saccade, a locus for fixation, or the reference for perceptual localization. We compared perceptual and saccadic localization for non-convex shapes where the center of gravity (COG) was located outside the boundary of the shape and did not coincide with any prominent perceptual features. The landing positions of single saccades made to the shape, as well as the preferred loci for fixation, were near the center of gravity, although local features such as part boundaries were influential. Perceptual alignment positions were also close to the center of gravity, but showed configural effects that did not influence either saccades or fixation. Saccades made in a more naturalistic sequential scanning task landed near the center of gravity with a considerably higher degree of accuracy (mean error <4% of saccade size) and showed no effects of local features, constituent parts, or stimulus configuration. We conclude that perceptual and oculomotor localization is based on the computation of a precise central reference position, which coincides with the center of gravity in sequential scanning. The saliency of the center of gravity, relative to other prominent visual features, can depend on the specific localization task or the relative configuration of elements. Sequential scanning, the more natural of the saccadic tasks, may provide a better way to evaluate the "default" reference position for localization. The fact that the reference position used in both oculomotor and perceptual tasks fell outside the boundary of the shapes supports the importance of spatial pooling, in contrast to local features, in object localization.     

Relation between complications of type I diabetes mellitus and collagen-linked fluorescence

Nonenzymatically glycosylated proteins gradually form fluorescent cross-linked protein adducts--a process termed "browning." The rate of this reaction increases with the glucose concentration. Assaying for the presence of browning products in long-lived proteins should therefore provide information on long-term metabolic control. We measured collagen-linked fluorescence typical for nonenzymatic browning in skin-biopsy specimens from 41 subjects with longstanding Type I diabetes and from 25 controls. Fluorescence correlated with age and (weakly) with the duration of diabetes. Mean age-adjusted fluorescence values were twice as high in diabetic subjects as in control subjects (P less than 0.0001) and increased with the severity of retinopathy, nephropathy, and arterial and joint stiffness. The correlation was significant for retinopathy (r = 0.42; P less than 0.01), arterial stiffness (r = 0.41; P less than 0.01), joint stiffness (r = 0.34; P less than 0.05), and the sum of all complications (r = 0.47; P less than 0.01). Fluorescence also correlated with systolic (r = 0.42; P less than 0.01) and diastolic (r = 0.36; P less than 0.05) blood pressures. If one can assume that the fluorescence results from a browning product of glucose, our data suggest that there is an overall correlation between the severity of diabetic complications and cumulative glycemia over many years.     

Substrate-specific regulation of the ribosome– translocon junction by N-terminal signal sequences

Amino-terminal signal sequences target nascent secretory and membrane proteins to the endoplasmic reticulum for translocation. Subsequent interactions between the signal sequence and components of the translocation machinery at the endoplasmic reticulum are thought to be important for the productive engagement of the translocon by the ribosome-nascent chain complex. However, it is not clear whether all signal sequences carry out these posttargeting steps identically, or if there are differences in the interactions directed by one signal sequence versus another. In this study, we find substantial differences in the ability of signal sequences from different substrates to mediate closure of the ribosome--translocon junction early in translocation. We also show that these differences in some cases necessitate functional coordination between the signal sequence and mature domain for faithful translocation. Accordingly, the translocation of some proteins is sensitive to replacement of their signal sequences. In a particularly dramatic example, the topology of the prion protein was found to depend highly on the choice of signal sequence used to direct its translocation. Taken together, our results reveal an unanticipated degree of substrate-specific functionality encoded in N-terminal signal sequences.     

Aleukemic leukemia cutis

Aleukemic leukemia cutis has always been a dermatological curiosity. It is a rare condition characterized by the infiltration of skin by leukemic cells before their appearance in the peripheral blood or bone marrow. A 20 year old man had presented with nodular swelling on the scalp of 6 months duration along with cervical lymphadenopathy. Biopsy and immunohistochemistry revealed myeloid sarcoma. The initial presentation was aleukemic and repeated peripheral blood counts and marrow examinations were normal. However, the outcome was fatal within 3 months of diagnosis of cutaneous lesions.