Localization of BiP to translating ribosomes increases soluble accumulation of secreted eukaryotic proteins in an Escherichia coli cell-free system

The endoplasmic reticulum (ER) resident Hsp70 chaperone, BiP, docks to the Sec translocon and interacts co-translationally with polypeptides entering the ER to encourage proper folding. In order to recreate this interaction in Escherichia coli cell-free protein synthesis (CFPS) reactions, a fusion protein was formed between the ribosome-binding portion of the E. coli protein trigger factor (TF) and BiP. The biophysical affinity to ribosomes as well as the characteristic Hsp70 ATPase activity were both verified for the fusion protein. When added to E. coli-based CFPS reactions, the TF-BiP fusion chaperone increased soluble yields of several protein fragments that are normally secreted through the ER and have poor solubility in typical CFPS reactions. For comparison, a fusion between TF and the native E. coli Hsp70, DnaK, was also constructed. This fusion was also biologically active and increased soluble yields of certain protein targets in CFPS. The TF-BiP fusion described in this study can be seen as a first step in reconstituting and better understanding ER folding pathways in the prokaryotic environment of E. coli CFPS.     

Harmonised Australian Reference Intervals for Serum PINP and CTX in Adults

Bone turnover markers (BTMs) are classified as either formation or resorption markers. Their concentrations in blood or urine of adults are considered to reflect the rate of bone remodelling and may be of use in the management of patients with bone disease. Major inter-method differences exist for BTMs, and harmonisation of methods is currently being pursued at an international level. Based on published data, this article describes age- and sex-specific Australian consensus reference intervals for adults for serum procollagen type I amino-terminal propeptide (s-PINP) and serum β-isomerised carboxy-terminal cross-linking telopeptide of type I collagen (s-CTX).     

RCPAQAP First Combined Measurement and Reference Interval Survey

Reference intervals are commonly considered to allow for between-laboratory bias. The RCPAQAP Liquid Serum Chemistry Program has collected data on laboratory measurements as well as reference intervals. This allows assessment of the between-laboratory variation in results, reference intervals and the information transmitted by the combination of these factors. For the majority of common chemistry analytes, the between-laboratory variation in reference intervals is greater than the variation in results. Additionally the reference interval variation is generally not related to bias between the results. Use of common reference intervals, either as an average of the current intervals in use, or the intervals proposed by the AACB Harmonisation Group, improved the variation seen in the information produced by different laboratories.     

Effect of Diet on Preference and Intake of Sucrose in Obese Prone and Resistant Rats

Increased orosensory stimulation from palatable diets and decreased feedback from gut signals have been proposed as contributing factors to obesity development. Whether altered taste functions associated with obesity are common traits or acquired deficits to environmental factors, such as a high-energy (HE)-diet, however, is not clear. To address this, we examined preference and sensitivity of increasing concentrations of sucrose solutions in rats prone (OP) and resistant (OR) to obesity during chow and HE feeding and measured lingual gene expression of the sweet taste receptor T1R3. When chow-fed, OP rats exhibited reduced preference and acceptance of dilute sucrose solutions, sham-fed less sucrose compared to OR rats, and had reduced lingual T1R3 gene expression. HE-feeding abrogated differences in sucrose preference and intake and lingual T1R3 expression between phenotypes. Despite similar sucrose intakes however, OP rats consumed significantly more total calories during 48-h two-bottle testing compared to OR rats. The results demonstrate that OP rats have an innate deficit for sweet taste detection, as illustrated by a reduction in sensitivity to sweets and reduced T1R3 gene expression; however their hyperphagia and subsequent obesity during HE-feeding is most likely not due to altered consumption of sweets.     

Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

Gastrointestinal dysfunction remains a major cause of morbidity and mortality. Indeed, gastrointestinal (GI) motility in health and disease remains an area of productive research with over 1,400 published animal studies in just the last 5 years. Numerous techniques have been developed for quantifying smooth muscle activity of the stomach, small intestine, and colon. In vitro and ex vivo techniques offer powerful tools for mechanistic studies of GI function, but outside the context of the integrated systems inherent to an intact organism. Typically, measuring in vivo smooth muscle contractions of the stomach has involved an anesthetized preparation coupled with the introduction of a surgically placed pressure sensor, a static pressure load such as a mildly inflated balloon or by distending the stomach with fluid under barostatically-controlled feedback. Yet many of these approaches present unique disadvantages regarding both the interpretation of results as well as applicability for in vivo use in conscious experimental animal models. The use of dual element strain gages that have been affixed to the serosal surface of the GI tract has offered numerous experimental advantages, which may continue to outweigh the disadvantages. Since these gages are not commercially available, this video presentation provides a detailed, step-by-step guide to the fabrication of the current design of these gages. The strain gage described in this protocol is a design for recording gastric motility in rats. This design has been modified for recording smooth muscle activity along the entire GI tract and requires only subtle variation in the overall fabrication. Representative data from the entire GI tract are included as well as discussion of analysis methods, data interpretation and presentation.     

Multiple mechanisms of target cell disintegration are employed in cytotoxicity reactions mediated by human natural killer cells

The rate of disintegration of target cells subsequent to lytic programming by human peripheral blood natural killer (NK) cells was investigated using a quantitative calcium pulse technique. The rate of this initial calcium-independent target cell disintegration was indicative of a first-order decay process for programmed target cells with a calculated half-life of less than 3 min. This initial, rapid disintegration phase was independent of the overall cytotoxic activity of the lymphocyte preparation tested. Moreover, initial rates of target cell disintegration were comparable for target cell lines that exhibit up to 6-fold differences in overall susceptibility to natural cytotoxicity. In these studies we also consistently observed very slow, calcium-independent disintegration of additional target cells following apparent completion of the rapid disintegration process. Using a 51Cr release assay and K-562 target cells, the kinetics of this slow disintegration process were examined and found to be similar for donors exhibiting up to a 2-fold difference in overall cytotoxic activity and independent of the concentration of programed target cells. Whereas the initial rapid disintegration mechanism was independent of temperature over the range of 10-37 degrees C, the slow disintegration mechanism exhibited a direct dependence on the incubation temperature. Furthermore, we observed that supernatants obtained after the termination of lytic programing by ethylene diaminetetraacetic acid could effect the slow lysis of fresh NK-susceptible target cell lines. These results support the utilization of at least two distinct mechanisms for target cell lysis by human NK cells.     

Expanding the horizons in treatment of severe peripheral vascular disease using microsurgical techniques

The use of microvascular tissue transfer as an adjunct to arterial reconstruction has begun to have a positive impact on limb salvage in patients with advanced arteriosclerosis and nonhealing ischemic wounds. However, many patients with severe peripheral vascular insufficiency not amenable to conventional arterial reconstructive procedures eventually require limb amputation. We have treated 12 patients with advanced peripheral vascular disease and nonhealing ischemic wounds by three different methods. These included distal bypass alone, distal bypass done in conjunction with free-tissue transfer, and free-tissue transfer alone. All bypass grafts were done to vessels at or below the ankle using a reversed saphenous vein. In each case, the distal anastomosis was performed, using the operating microscope and standard microvascular technique. Mean follow-up for these patients is 18 months. Distal bypass alone resulted in limb salvage in three of five patients. In the combined bypass and free-flap group, three of five patients had salvage of their threatened extremity at a 1-year follow-up. Two patients with ischemic ulcers, rest pain, and unsuitable distal vessels for bypass were treated with free-tissue transfer alone. This resulted in healed wounds, limb salvage, and complete resolution of the rest pain symptoms in both patients. When advanced ischemia is complicated by large areas of tissue loss, combined bypass and microvascular free-issue transfer, performed in stages or simultaneously, is safe and can often result in limb salvage. In the rare instance of a completely obliterated distal runoff bed, free-tissue transfer alone may provide not only a healed wound, but also a means of "indirect" revascularization of the extremity and limb salvage.     

Reconstruction of the cervical esophagus: free jejunal transfer versus gastric pull-up

Use of enteric grafts is a popular method for reconstruction of the cervical esophagus and hypopharynx. Free jejunal transfer (FJT) and gastric pull-up (GP) are the most popular methods used. This discussion is a retrospective review of our experience with 50 cases of free jejunal transfer and 15 cases of gastric pull-up. The graft survival rate was 94 percent (47 of 50) for free jejunal transfer and 87 percent (13 of 15) for gastric pull-up. Successful swallowing was achieved in 88 percent (44 of 50) of free jejunal transfers and 87 percent (13 of 15) of gastric pull-ups. Patients with free jejunal transfers were able to swallow and leave the hospital sooner: 10.6 versus 16.0 days and 22.3 versus 29.0 days, respectively. Fistulas occurred in 16 percent (8 of 50) of free jejunal transfers, most of which (6 of 8) healed spontaneously. Fistulas occurred in 20 percent (3 of 15) of gastric pull-ups, only one of which healed spontaneously. Stricture was the most common late complication for free jejunal transfers, 22 percent (11 of 50), whereas reflux was most common in gastric pull-ups, 20 percent (3 of 15). In patients with advanced cancer, extensive esophageal resection into the chest is often required, and gastric pull-up seems to be an easier and more direct form of reconstruction. In limited resection of the hypopharynx and esophagus, especially with proximal lesions, free jejunal transfer is simpler and avoids mediastinal dissection. This concept as well as other advantages and disadvantages of both techniques will be discussed.     

Natural occurrence of deoxynivalenol, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol,nivalenol, 4,7-dideoxynivalenol,and zearalenone in polish wheat

Three wheat samples collected in 1987 in Central Poland and naturally infected withFusarium spp were analyzed for the presence ofFusarium spp andFusarium toxins. Heads were separated into three fractions: kernels with visibleFusarium damage, healthy looking kernels, and chaff + rachis. The samples contained deoxynivalenol (2.0 – 40.0μg/g), nivalenol (O.O1μg/g), 4,7-dideoxynivalenol (0.10 – 0.15μg/g). 15-acetyldeoxynivalenol (0.10–2.00 μg/g), 3-acetyldeoxynivalenol (O/1Oμg/g), and zearalenone (0.01–2.00μg/g). This is the first report about 15 - acetyldeoxynivalenol in European wheat and the co-occurrence of 3 - acetyldeoxynivalenol and 15-acetyldeoxynivalenol in the same sample of contaminated cereals.     

The role of free-tissue transfers in lower-extremity reconstruction

Eighty-five free flaps were performed in 76 patients for defects in the lower extremity. A new classification of lower-extremity defects was devised to help define the role of free-tissue transfers: group 1, soft-tissue defects; group 2, soft-tissue and bone loss less than 8 cm; group 3, massive soft-tissue and bone loss greater than 8 cm; and group 4, bone defect only. Each group was further divided into clean (A) and infected (B) wounds. Our overall results include resolution of the presenting problem in 82 percent; there were 17 flap losses (20 percent), persistent osteomyelitis in 8, and 10 amputations. This review has prompted us to limit our indications for limb salvage, particularly in group 3B, in patients with compound injuries that include loss of plantar sensation, and in patients with large segments of infected bone.