Cellulose-bound Peptide Arrays: Preparation and Applications

Spatial organization of metabolic enzymes may represent a general cellular mechanism to regulate metabolic flux. One recent example of this type of cellular phenomenon is the purinosome, a newly discovered multi-enzyme metabolic assembly that includes all of the enzymes within the de novo purine biosynthetic pathway. Our understanding of the components and regulation of purinosomes has significantly grown in recent years. This paper reviews the purine de novo biosynthesis pathway and its regulation, and presents the evidence supporting the purinosome assembly and disassembly processes under the control of G-protein-coupled receptor (GPCR) signaling. This paper also discusses the implications of purinosome and GPCR regulation in drug discovery.     

Effect of unilateral partial facial paralysis on periosteal growth at the muscle-bone interface of facial muscles and facial bones

In a previous study, the influence of the midfacial musculature upon growth and development of the maxilla and mandible was established macroscopically. Dry skull measurements revealed a reduced premaxillary, maxillary, mandibular, and anterior corpus length with a simultaneous increase in mandibular ramal height on the paralyzed side. It was demonstrated that these reduced premaxillary and maxillary lengths were among others the result of reduced nasofrontal growth, whereas the increased ramal height was accompanied by condylar growth alterations. This study investigated whether the growth alterations at the mandibular corpus region could be explained by altered periosteal growth at the muscle-bone interface of the zygomatico-auricular muscle and the mandibular corpus, caused by altered muscle activity acting upon the periosteal sleeve. Fifty-six 12-day-old New Zealand White rabbits were randomly assigned to either a control or an experimental group. In the experimental group, left-sided partial facial paralysis was induced surgically when the animals were 12 days old. To study the muscle-bone interface, seven follow-up time intervals were defined between 3.5 and 60 days following the surgery. At these time intervals, four randomly selected control animals and four randomly selected experimental animals were killed. The anterior mandibular corpus region with the muscle-bone interface of the left control hemimandible and the left and right experimental hemimandibles was processed for undecalcified tissue preparation. Quantitative analysis of the total bone area at the muscle-bone interface revealed no significant differences between the left control hemimandible and the left and right experimental hemimandibles. Also, qualitative study of the histologic sections showed no major changes in the appearance or development of the trabecular pattern between the groups. However, slight differences in the distribution pattern of osteoblasts and osteoclasts along the bony surface were found between the left control hemimandible and the left and right experimental hemimandibles, which seemed to explain the alterations in mandibular corpus shape between these groups. It was suggested that these changes in the distribution pattern of osteoblasts and osteoclasts were the result of changes in the loading distribution pattern acting upon the mandible, caused by an altered neuromuscular recruitment pattern of the remaining functionally intact, mandibularly attached muscles. The latter was probably the result of adaptive mandibular positioning in response to an altered occlusal relationship, which was induced by the abnormal maxillary growth as a result of the unilateral partial facial paralysis.     

Facial contour restoration in Barraquer-Simons syndrome using two free anterolateral thigh flaps

Barraquer-Simons syndrome, or cephalothoracic lipodystrophy, is characterized by fat atrophy of an obscure pathogenesis involving the face and, eventually, the thoracic region. Simultaneously, fat hypertrophy of the lower extremities, a nephropathy, and complement anomalies may be observed. We presented two patients with the typical features of this disease, as well as a previously undescribed vascular and perivascular inflammation of the facial arteries and veins that caused problems with microvascular anastomosis. Both patients were treated with a bilateral transfer of the anterolateral thigh flap, which has not been reported previously. In contrast to other transfers previously reported, the fat tissue of this flap is never affected by the disease and is redundantly present. Placing the fascia of the flaps toward the skin allows for strong fixation to the temporal region and guarantees a stable result with a smooth facial contour.     

The effect of muscle transplantation after unilateral partial facial paralysis on craniofacial growth and development: relationship between muscle and nerve histomorphometric findings

Muscle transplantation has become an indispensable tool to restore the smile in patients with long-standing or congenital facial paralysis. However, little is known of the effect of this surgical intervention on craniofacial growth and development or of the adaptation of the transplant to its recipient site under circumstances of growth. The present study investigates these phenomena in the rabbit model. Twelve-day-old New Zealand White rabbits were randomly assigned to three experimental groups. The control group was used to study normal craniofacial growth and development (n = 15). In the nerve ablation group, unilateral paralysis of the buccal branches of the facial nerve was surgically induced (n = 15). In the transplant group, the surgically induced unilateral paralysis of the buccal branches was immediately followed by a neuromuscular graft (n = 12). All animals were operated on at the age of 12 days, and follow-up evaluations were performed at the ages of 2 months and 6 months. Computerized dorsoventral roentgencephalometric and computed tomography investigations were performed at both ages. Nerve and muscle histomorphometric measurements were performed at the age of 6 months to relate the quality of nerve and muscle regeneration to the growth parameters. The roentgencephalometric measurements revealed that analogous disturbed parameters were present in the nerve ablation and the transplant groups. However, in the transplant group, an additional significant effect of time between 2 and 6 months was seen for some parameters. This resulted in significant differences between the nerve ablation and transplant groups at 6 months for these parameters. Computed tomography measurements showed no significant differences in maxillary or mandibular volume in the transplant group compared with the control or nerve ablation groups. However, a significantly diminished increase in bone volume existed in the transplant group for the time period between 2 and 6 months in comparison with the control and nerve ablation groups. Muscle histomorphometric findings revealed a significant change in muscle fiber composition in the graft compared with the normal latissimus dorsi muscle; this was due to a major decrease in type IIB fibers, with an increase in type I and type IIA fibers. Compared with the normal zygomaticoauricular muscle, the amount of type I fibers was significantly increased. No fiber atrophy was found. Macroscopically, the transplanted muscle failed to increase its length during growth. Nerve histomorphometric findings demonstrated a normal amount of nerve fibers; however, they had significantly decreased diameters and reduced myelin areas. The nerve histomorphometric parameters were related to the muscle histomorphometric findings, which in turn were related to craniofacial growth disturbances. These findings suggested that the main growth differences between the transplant group and the control group may have been due to altered nerve function influencing muscle function. Scar tissue formation and the development of more intense muscle activity later are suggested as the causes of the additional effect of time between 2 and 6 months for the several parameters in the transplant group. Reasons for the failure of complete conversion of the graft to a fast muscle and the failure of the transplant to elongate during growth are discussed.     

One-step refolding and purification of disulfide-containing proteins with a C-terminal MESNA thioester

Background  

Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed.     

Kinematic performance of a six degree-of-freedom hand model (6DHand) for use in occupational biomechanics

Upper extremity musculoskeletal disorders represent an important health issue across all industry sectors; as such, the need exists to develop models of the hand that provide comprehensive biomechanics during occupational tasks. Previous optical motion capture studies used a single marker on the dorsal aspect of finger joints, allowing calculation of one and two degree-of-freedom (DOF) joint angles; additional algorithms were needed to define joint centers and the palmar surface of fingers. We developed a 6DOF model (6DHand) to obtain unconstrained kinematics of finger segments, modeled as frusta of right circular cones that approximate the palmar surface. To evaluate kinematic performance, twenty subjects gripped a cylindrical handle as a surrogate for a powered hand tool. We hypothesized that accessory motions (metacarpophalangeal pronation/supination; proximal and distal interphalangeal radial/ulnar deviation and pronation/supination; all joint translations) would be small (less than 5° rotations, less than 2mm translations) if segment anatomical reference frames were aligned correctly, and skin movement artifacts were negligible. For the gripping task, 93 of 112 accessory motions were small by our definition, suggesting this 6DOF approach appropriately models joints of the fingers. Metacarpophalangeal supination was larger than expected (approximately 10°), and may be adjusted through local reference frame optimization procedures previously developed for knee kinematics in gait analysis. Proximal translations at the metacarpophalangeal joints (approximately 10mm) were explained by skin movement across the metacarpals, but would not corrupt inverse dynamics calculated for the phalanges. We assessed performance in this study; a more rigorous validation would likely require medical imaging.     

Analysis of the musculoskeletal loading of the thumb during pipetting – A pilot study

Previous epidemiological studies indicate that the use of thumb-push mechanical pipettes is associated with musculoskeletal disorders (MSDs) in the hand. The goal of the current study was to analyze the loading in the muscle–tendon units in the thumb during pipetting. The hand is modeled as a multi-body linkage system and includes four fingers (index, long, ring, and little finger), a thumb, and a palm segment. Since the current study is focused on the thumb, the model includes only nine muscles attached to the thumb via tendons. The time-histories of joint angles and push force at the pipette plunger during pipetting were determined experimentally and used as model input; whereas forces in the muscle–tendon units in the thumb were calculated via an inverse dynamic approach combined with an optimization procedure. Results indicate that all nine muscles have force outputs during pipetting, and the maximal force was in the abductor pollicis brevis (APB). The ratio of the mean peak muscle force to the mean peak push force during the dispensing cycle was approximately 2.3, which is comparable to values observed in grasping tasks in the literature. The analysis method and results in the current study provide a mechanistic understanding of MSD risk factors associated with pipetting, and may be useful in guiding ergonomic designs for manual pipettes.     

Ca2+ dependence of transverse tubule-mediated calcium release in skinned skeletal muscle fibers

Isometric force and 45Ca efflux from the sarcoplasmic reticulum were measured at 19 degrees C in frog skeletal muscle fibers skinned by microdissection. After Ca2+ loading, application of the ionophores monensin, an Na+(K+)/H+ exchanger, or gramicidin D, an H+ greater than K+ greater than Na+ channel-former, evoked rapid force development and stimulated release of approximately 30% of the accumulated 45Ca within 1 min, whereas CCCP (carbonyl cyanide pyruvate p-trichloromethoxyphenylhydrazone), a protonophore, and valinomycin, a neutral, K+-specific ionophore, did not. When monensin was present in all bathing solutions, i.e., before and during Ca2+ loading, subsequent application failed to elicit force development and to stimulate 45Ca efflux. 5 min pretreatment of the skinned fibers with 50 microM digitoxin, a permeant glycoside that specifically inhibits the Na+,K+ pump, inhibited monensin and gramicidin D stimulation of 45Ca efflux; similar pretreatment with 100 microM ouabain, an impermeant glycoside, was ineffective. Monensin stimulation of 45Ca efflux was abolished by brief pretreatment with 5 mM EGTA, which chelates myofilament-space calcium. These results suggest that: monensin and gramicidin D stimulate Ca2+ release from the sarcoplasmic reticulum that is mediated by depolarization of the transverse tubules, which seal off after sarcolemma removal and form closed compartments; a transverse tubule membrane potential (myofilament space-negative) is maintained and/or established by the operation of the Na+,K+ pump in the transverse tubule membranes and is sensitive to the permeant inhibitor digitoxin; the transverse tubule-mediated stimulation of 45Ca efflux appears to be entirely Ca2+ dependent.