Regulation of leptin, adiponectin and acylation-stimulating protein by hyperinsulinaemia and hyperglycaemia in vivo in healthy lean young men.

AIM: Both type 1 and 2 diabetes are associated with differential regulation of leptin, adiponectin and ASP. Our aim was to examine whether or not acute hyperinsulinaemia and/or hyperglycaemia per se have differential regulation of these hormones in healthy subjects. METHODS: We examined changes in leptin, adiponectin and ASP concentrations and subcutaneous white adipose tissue mRNA expression with 3-hour hyperinsulinaemic (HI, n=10), hyperglycaemic (HG, n=7) and hyperinsulinaemic-hyperglycaemic (HGHI, n=8) clamps in healthy lean young men. As somatostatin was used for the HG and HGHI clamps, a control somatostatin clamp was carried out (n=4). Changes in the expression of HKII and p85alpha Pi3K were examined as positive controls for the induction of gene expression by the insulin pathway. RESULTS: HI, HG and HGHI clamps increased expression of HKII and p85alpha Pi3K while somatostatin did not. The HI clamp decreased serum adiponectin (-15%, P<0.001) and increased serum leptin (+11%, P=0.031), while the HG clamp reduced serum leptin (-20%, P=0.003). The HGHI clamp increased serum ASP (+21%, P=0.047) and expression of C3 (+26%, P=0.018) and leptin (+50%, P=0.024). Interestingly, the control somatostatin clamp suppressed both serum leptin (-17%, P=0.043) and adiponectin (-7%, P=0.020). CONCLUSION: HG and/or HI per se regulated the concentrations and expression of leptin, adiponectin and ASP in healthy lean young men, suggesting a contribution to dysregulation of these hormones in diabetes.

Detection of rib trauma in newborn foals in an equine critical care unit: a comparison of ultrasonography, radiography and physical examination.

REASONS FOR PERFORMING STUDY: Previous studies have shown that in man ultrasonography is more accurate than radiography for detecting rib fractures. OBJECTIVES: To describe clinical, radiographic and ultrasonographic findings related with rib fractures in newborn foals in an equine critical care unit; and to compare diagnostic accuracy of ultrasonography to radiography. METHODS: A prospective ultrasonographic study was performed on 29 foals presented to the emergency unit. This study was performed at the Centre Hospitalier Universitaire Vétérinaire (CHUV), University of Montreal. Physical examination as well as radiographic and ultrasonographic examinations were performed. RESULTS: Thoracic radiographs revealed 10 rib fractures in 5 of 26 (19%) foals. Ultrasonography revealed 49 fractures in 19 of 29 (65%) foals of which fillies (n = 13; 68%) were significantly over represented as were fractures to the left thorax (n = 15; 78%). Seventeen of 19 foals (90%) had rib fractures located 3 cm or less from the costochondral junction, the distal part of the rib being displaced laterally in all cases. In 2 foals, where both thoracic radiographs and ultrasonography detected rib fractures, the site of fractures was located on the mid portion of the rib. Rib fractures were detected only by thoracic radiographs in one foal. Sixty-five percent (32/49) of fractured ribs had a moderate displacement (1-4 mm). CONCLUSIONS: Rib fractures are seen frequently in newborn foals in equine critical care units. Ultrasonography is more accurate than radiography and reveals fractures in most patients presented in emergency. The position (costochondral junction) of rib fractures and of the fragments suggest that most thoracic trauma probably occurs during parturition. POTENTIAL RELEVANCE: Ultrasound imaging increases awareness and improves the diagnosis of rib fractures in newborn foals.

Pharmacokinetics of morphine and its glucuronidated metabolites in burn injuries.

OBJECTIVE: To investigate the effects of major thermal burn injury and continuous intravenous morphine infusion on the disposition of morphine and its glucuronidated metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) once a week for three weeks. CASE SUMMARIES: Five patients with major first-, second-, or third-degree burn injuries received long-term intravenous morphine infusion. The required dose varied greatly (from 4 to 39.5 mg/h). The steady-state concentrations of morphine, M3G, and M6G ranged from 20 to 452, 29 to 3436, and 20 to 1240 mumol/L, respectively. The systemic clearance (Cls) of morphine ranged from 14.8 to 40.3 mL/min/kg and did not change over time. The ratios of M6G and M3G to morphine were not affected by dose, even with the wide variation of intravenous dosage. Morphine kinetics appeared to be first-order. Mean recovery of morphine, M3G, and M6G in urine was 1.7 +/- 1.0%, 42.0 +/- 16.8%, and 11.8 +/- 3.2%, respectively, and renal clearance ranged from 8 to 64, 26 to 325, and 59 to 589 mL/min, respectively. Mean pain intensity ratings at rest remained low and stable (0.7 +/- 0.9 on day 7, 0.4 +/- 0.3 on day 14, 0 +/- 0 on day 21). DISCUSSION: To our knowledge, this is the first published report describing morphine, M3G, and M6G disposition in patients with major thermal burn injury. The Cls of morphine is similar to that observed in other patient populations and healthy subjects, suggesting that the presence of major burn injuries or a continuous morphine infusion over a three-week period may not contribute significantly to the variability among individuals. In these cases, the renal clearance of morphine and its glucuronides was within the range of values reported for other populations of patients and healthy subjects. Recovery of morphine and its glucuronides in urine was also similar to that in healthy individuals. CONCLUSIONS: These cases suggest that the effects of major burn injuries and of long-term intravenous infusion of morphine did not seem to modify morphine, M3G, and M6G disposition. Among patients with burn injuries, the severity of burns of duration of administration are not a cause of nonlinear kinetic of morphine or of morphine resistance. The morphine infusion rate was substantially variable and not directly related to its clearance, suggesting that monitoring of morphine should be focused on the clinical response.

The intellectual property cookbook: a guide for the novice health-care telemedicine provider working with industry.

Telemedicine is a new field and many health-care providers are developing their own products with the help of industry. Most practitioners are novices in the legal tools necessary to protect their own work with regard to any future commercialization. To summarize these issues for the telemedicine practitioner, a review of intellectual property protection has been performed. Intellectual property can be protected by tools such as copyrights, patents, non-disclosure and confidentiality agreements, integrated circuit topographies and industrial design. Knowledge of the intellectual property background should allow telemedicine providers to protect their own work when working with industry.

Surgical treatment of lumbosacral instability caused by discospondylitis in four dogs.

OBJECTIVE: To describe a surgical technique involving distraction and stabilization of the lumbo-sacral vertebral segment using an external skeletal fixator in dogs with lumbosacral instability caused by discospondylitis. STUDY DESIGN: Retrospective clinical study. ANIMALS: Four client-owned dogs. METHODS: Medical records of all dogs diagnosed with discospondylitis from 1994 to 1997 were identified and reviewed. Four dogs with lumbosacral discospondylitis requiring surgical treatment were then specifically studied. Surgical technique, clinical signs, preoperative diagnostic investigation, radiographic findings, and the results of short-term and long-term reevaluations were recorded. RESULTS: Twelve dogs with discospondylitis were identified, 4 of which had lumbosacral discospondylitis. These 4 dogs underwent surgical distraction and stabilization because they failed to respond to medical treatment. Three dogs received a cancellous bone graft between L7 and S1 and had rapid interbody fusion of this vertebral segment. The dog that did not receive a graft did not have interbody fusion at the time of fixator removal. This did not affect the final clinical outcome. Lumbosacral pain and neurological deficits present before surgery rapidly subsided after the procedure. All dogs received concurrent antibiotic treatment for a minimum of 4 weeks. All dogs were clinically normal at the time of fixator removal and all continued to do well during the follow-up period (8-48 months; mean, 27.5 months). CONCLUSION AND CLINICAL RELEVANCE: Lumbosacral discospondylitis may not respond well to conservative treatment because of the mobility of the affected space. Surgical treatment involving distraction and stabilization to obtain intervertebral fusion is very effective in treating lumbosacral instability caused by discospondylitis.

Clinical wear of 63 ultrahigh molecular weight polyethylene acetabular components: effect of starting resin and forming method.

This study compares the clinical wear rates and implant characteristics of 63 surgically retrieved acetabular components. All components were made by the same manufacturer, implanted by the same surgeon, in articulation against the same type of femoral component, and revised for the same reason; 19 were made from directly compression molded, calcium stearate free, ultrahigh molecular weight polyethylene (UHMWPE) and 44 were made from machined, ram extruded, calcium stearate containing UHMWPE. There were significant differences in wear, duration, and wear rate between the molded (type I) and machined (type II and III) components. Most importantly, the wear rates of type I (molded) components were significantly (p < 0.0001) lower than the wear rates of type II, type III, and type II and III components as a group (all machined). The machined components had wear rates 2.3 times greater than the molded components. The wear rates between the two different groups of machined components (type II and III) were not significantly different. The scanning electron microscope observations did not reveal any major differences in wear mechanisms between the three types of components, although the machined components did show more evidence of brittleness. The molded components were better consolidated (or had less fusion defects) than the ram extruded components.

Synthesis and characterization of a novel UHMWPE interpenetrating polymer network.

A novel, biomimetic, interpenetrating polymer network (IPN) between poly-L-lysine (PLL) and ultra high molecular weight polyethylene (UHMWPE) has been synthesized in an attempt to decrease wear in joint prostheses. A biomaterial with a gradient IPN of cationic PLL and UHMWPE has been synthesized, in the surface of bulk UHMWPE, to recruit the poly-anion, hyaluronic acid, from the synovial fluid. It is hypothesized that the hyaluronic acid molecules and their associated hydration layer will improve lubrication between the articulating surfaces, thus lowering both friction and wear. The synthesis involves four steps. Silylation of the PLL-HBr to PLL-SiMe3 utilizing bis(trimethylsiyl)acetamide (BSA). Swelling of the UHMWPE in a solution of PLL-SiMe3/xylenes at 60 degrees C with ultrasonics. Crosslinking of the PLL-SiMe3 within the UHMWPE with 1,8-diisocyanatooctane (a.k.a. OMDI). Finally, de-swelling and drying of the IPN under vacuum at 50 C. Visual observations show an adhered film on the IPN surface. Reflective FTIR spectra contain the characteristic peaks associated with UHMWPE. Two additional peaks, at 3410 and 1690 cm-1, are associated with PLL. SEM shows a morphology dominated by PLL spheres with diameters ranging from < 1 micron up to 3 micron. This shows that the PLL-SiMe3 has been crosslinked by the OMDI and was not rinsed away by either xylenes or sonicated water rinses. High contact angle of the PLL in contact with the UHMWPE demonstrate that the PLL has been de-silylated and returned to its hydrophilic nature. The spheres attached to the surface of the UHMWPE indicate that PLL has infiltrated the UHMWPE physical network and is entangled there. XPS confirms the presence of nitrogen by a 3-5 atomic percent signal in the outer 100 A of the IPN surface.

Comparison of ketoprofen, oxymorphone hydrochloride, and butorphanol in the treatment of postoperative pain in dogs.

OBJECTIVE: To compare analgesic effects of ketoprofen, oxymorphone hydrochloride, and butorphanol when used to control postoperative pain associated with elective orthopedic surgery in dogs. DESIGN: Prospective randomized clinical trial. ANIMALS: 70 dogs undergoing orthopedic surgery on a hind limb. PROCEDURE: Dogs were randomly assigned to 1 of 4 postoperative analgesic treatment groups: ketoprofen alone, oxymorphone alone, butorphanol alone, or ketoprofen-oxymorphone. Drugs were given IM at the end of anesthesia. Pain score, sedation score, arterial blood pressures, arterial blood gas partial pressures, and plasma cortisol concentration were measured for 12 hours after surgery. If the pain score was > or = 9, supplemental oxymorphone was administered IM. RESULTS: The proportion of dogs that did not require supplemental treatment with oxymorphone was significantly higher for the ketoprofen alone and ketoprofen-oxymorphone groups than for the oxymorphone alone group. During the first hour after surgery, pain score was lower for oxymorphone alone and ketoprofen-oxymorphone groups than for ketoprofen or butorphanol alone groups. Significant differences were not detected among groups in regard to pain score 2 and 3 hours after surgery or in regard to arterial blood pressures at any time. From 4 to 12 hours after surgery, pain score was significantly lower for the ketoprofen alone group than for other groups. Plasma cortisol concentration was significantly higher for the oxymorphone alone group 6 and 8 hours after surgery, compared with other groups. CLINICAL IMPLICATIONS: Except during the first hour after surgery, dogs given ketoprofen alone after elective orthopedic surgery had a greater level of, and longer-lasting, analgesia than did dogs given oxymorphone or butorphanol alone.

Malignant giant cell tumour of the distal femur treated by excision, allografting and ligamentous reconstruction: an 18-year follow-up.

Extensive osteoarticular allografts have been used for knee reconstruction, but because of their composite nature and the technical difficulty of the procedure, complication and failure rates have been high. There are few records of long-term results in the literature. In this report, a 19-year-old man with a large aggressive giant cell tumour of the left distal femur was treated in 1976 by en bloc resection, massive femoral allografting and ligamentous reconstruction. Follow-up after 18 years showed no recurrence of the tumour, excellent incorporation of the graft and good knee function, which allowed the patient to work 9 hours a day on his feet without pain.

Comparison of the in vivo wear rates of 43 surgically retrieved direct compression molded and ram extruded ultra high molecular weight polyethylene acetabular components.

The objective of this study was to investigate retrieved ultra high molecular weight polyethylene (UHMWPE) liners from Total HIp Arthroplasty (THA) revisions. The amount of in vivo wear on compression molded v. ram extruded and machined acetabular liners was the focus of comparison between 43 surgically retrieved samples. The principal experimental methods were scanning electron microscope (SEM) observations and a fluid displacement method to quantify volumetric wear. SEM observations revealed similar wear mechanisms between the compression molded and ram extruded and machined UHMWPE retrieved liners. More third body wear corresponded with more volumetric in vivo wear. This study found that the ram extruded and machined UHMWPE liners had a significantly larger average wear rate than the compression molded UHMWPE liners. In order to determine whether the observed difference in wear rates can be attributed to the different types of UHMWPE processing, a more controlled experiment would have to be performed.

Radiation inactivation and in situ renaturation of protein tyrosine kinases reveal a major 50-kDa enzyme as part of a membrane complex present in dividing but not in resting prostatic epithelial cells.

Because protein tyrosine kinases play a crucial role in the regulation of cell division and carcinogenesis, we have herein measured such enzyme activities (specific activity and subcellular distribution) and compared their characteristics with respect to hydrodynamic properties and radiation inactivation sizes as well as renaturation after electrophoresis in denaturing conditions in canine prostatic epithelial cells either in a resting (freshly isolated) or in a dividing (cultured cells) state. In quiescent cells, most protein tyrosine kinase activity was expressed by soluble proteins with a Stokes' radius (Rs) of 3.05 nm, a sedimentation coefficient (S20,w) of 4.0 S, and a molecular mass of 50 kDa. By contrast, in dividing cells (three days in primary culture), the specific activity was higher and the enzyme was mainly membrane bound. The use of a detergent (Triton X-100) allowed the extraction of most of that enzyme; its partial specific volume, S20,w and Rs were then 0.883 cm3/g, 4.0 S, and 5.6 nm, respectively, hence yielding a molecular mass of 215 kDa, which decreased to 125-145 kDa when corrected for detergent binding. Probing these chromatography-peak fractions, 50 kDa from cytosol of resting cells and 215 kDa from membrane extracts of dividing cells, with a phosphotyrosine antibody following their incubation with ATP and electrophoresis in denaturing conditions revealed the presence of a common 50-kDa phosphotyrosylated protein along with three other bands (130, 75, and 40 kDa) in the high-Mr peak of enzyme. However, the radiation inactivation size for protein tyrosine kinases expressed in both resting and dividing cells were similar, 47.2 +/- 8.7 and 44.5 +/- 6.1 kDa, respectively. Furthermore, by renaturation after electrophoresis in denaturing conditions, major protein tyrosine kinase polypeptides of 50 kDa were identified in both cell populations. Taken together, these results indicate that, in dividing prostatic epithelial cells, membrane-bound protein tyrosine kinases of low molecular weight with properties similar to those of monomeric soluble forms present in quiescent cells are part of high-molecular weight complexes. This activation process may be critical for hormone-independent proliferation of prostatic epithelial cells.

Manual discrimination of compliance using active pinch grasp: the roles of force and work cues.

In these experiments, two plates were grasped between the thumb and the index finger and squeezed together along a linear track. The force resisting the squeeze, produced by an electromechanical system under computer control, was programmed to be either constant (in the case of the force discrimination experiments) or linearly increasing (in the case of the compliance discrimination experiments) over the squeezing displacement. After completing a set of basic psychophysical experiments on compliance resolution (Experiment 1), we performed further experiments to investigate whether work and/or terminal-force cues played a role in compliance discrimination. In Experiment 2, compliance and force discrimination experiments were conducted with a roving-displacement paradigm to dissociate work cues (and terminal-force cues for the compliance experiments) from compliance and force cues, respectively. The effect of trial-by-trial feedback on response strategy was also investigated. In Experiment 3, compliance discrimination experiments were conducted with work cues totally eliminated and terminal-force cues greatly reduced. Our results suggest that people tend to use mechanical work and force cues for compliance discrimination. When work and terminal-force cues were dissociated from compliance cues, compliance resolution was poor (22%) relative to force and length resolution. When work cues were totally eliminated, performance could be predicted from terminal-force cues. A parsimonious description of all data from the compliance experiments is that subjects discriminated compliance on the basis of terminal force.

Ultrasonography of the ankle and the hindfoot.

High-resolution ultrasonography (US) has proved an excellent noninvasive and inexpensive modality for examining the extremities. This pictorial essay illustrates the US appearance of bony and soft-tissue abnormalities of the ankle and the hindfoot. The role of US as an adjunct technique to complement conventional radiography is stressed. The advantages of US in examining traumatic, inflammatory and infectious lesions and soft-tissue masses are discussed.

Scintigraphic "doughnut sign" on skeletal imaging due to a hemangioendothelioma of bone.

A 61-yr-old patient was referred to our hospital for investigation of pain and tenderness in his left lower limb. Bone scan revealed multiple lesions of tibia and foot, several of which appeared as doughnut-like lesions and corresponded to lytic abnormalities on radiographs. Pathologic examination revealed multiple epithelioid hemangioendothelioma of bone.

Inactivation mechanism of tetrameric beta-galactosidase by gamma-rays involves both fragmentation and temperature-dependent denaturation of protomers.

The radiation inactivation method is widely used to estimate the molecular size of membrane-bound enzymes, receptors, and transport systems in situ. The method is based on the principle that exposure of frozen solutions or lyophilized protein preparations to increasing doses of ionizing radiations results in a first-order decay of biological activity proportional to radiation inactivation size of the protein. This parameter is believed to reflect the "functional unit" of the protein defined as the minimal assembly of structure (protomers) required for expression of a given biological activity. We tested the functional unit as a concept to interpret radiation inactivation data of proteins with Escherichia coli beta-galactosidase, where the protomers are active only when associated in a tetramer. Gamma-Irradiation of beta-galactosidase at both -78 and 38 degrees C followed by quantitation of the residual unfragmented promoter band by SDS-polyacrylamide gel electrophoresis yielded the protomer size, indicating that only one protomer is fragmented by each radiation hit. By following the enzyme activity as a function of dose it was found that only the protomer that has been directly hit and fragmented at -78 degrees C was effectively inactivated. In contrast, at 38 degrees C, it was the whole tetramer that was inactivated. beta-Galactosidase cannot have two different functional units depending on temperature. The inactivation of the whole beta-galactosidase tetramer at 38 degrees C is in fact related to protomer fragmentation but also to the production of stable denatured protomers (detected by gel-filtration HPLC and differential UV spectroscopy) due to energy transfer from fragmented protomers toward unhit protomers.(ABSTRACT TRUNCATED AT 250 WORDS)

Radiation inactivation of estrogen receptor in intact human breast cancer cells (MCF-7). Lack of energy transfer to the estradiol binding domain.

Whole MCF-7 human breast-cancer cells were irradiated at - 78 degrees C in a calibrated Gammacel 60Co irradiator. Freezing or storing conditions induce neither an alteration of the viability of cells nor a change in estradiol binding activity. Hexosaminidase was used as internal marker, and we measured the radiation inactivation size (RIS) of the estrogen receptor in whole cells. After various cell treatments, the estradiol binding unit always presents a molecular mass of 25 kDa. This value, which corresponds to the size of the defined hormone binding domain of the estrogen receptor, suggests that the energy delivered to the protein by the radiation is efficient to inactivate estradiol binding only when the hit occurs directly in the smaller hormone binding domain.

Radiation-inactivation size of transformed and non-transformed androgen receptors.

The nucleic acid sequence of the androgen receptor (AR) gene predicts that the protein structure possesses DNA- and steroid-binding domains that show high degrees of sequence similarity with those of other steroid receptors. Since the steroid-binding domain of the AR corresponds to a 30 kDa portion of the protein, and the AR structure may be monomeric or hetero-oligomeric depending on its transformation state, we have herein determined the AR radiation-inactivation size (RIS) in relation to the molecular structure whose binding activity toward methyltrienolone (R1881) is abolished by a radiation 'hit'. Soluble fractions from whole canine prostatic tissue were used as a source of non-transformed AR. The AR transformation was induced by the addition of 0.6 M-KCl, and these preparations were used together with high-salt nuclear extracts as a source of transformed AR. To maximize the binding activity, molybdate and dithiothreitol were included during AR extraction. Receptor transformation was verified by modifications of both the sedimentation coefficients (from 7.5 S to 4.1 S on sucrose gradients) and molecular masses (from 260 kDa to 115 kDa by gel filtration). The RIS values of the non-transformed and transformed ARs were not statistically different: 92 +/- 19 kDa and 110 +/- 25 kDa respectively. In addition, the inactivation of AR binding activity by radiation was attributed to a loss of binding sites, with no significant change in the Kd. When benzoic acid, a free-electron scavenger, was added together with dithiothreitol before and after irradiation, no change in the RIS value was observed. Thus, in the canine prostate, the RIS value of the AR represents the monomeric protein, independently of its association with other proteins, and this value corresponds to that predicted by cloning studies and photoaffinity-labelling of AR.

Target size analysis of estrogen receptor in cultured intact cells: change in receptor structure between subconfluency and superconfluency in culture.

MCF-7 human breast cancer cells were submitted to the tritiated antiestrogen tamoxifen aziridine, frozen at -170 degrees C, stored and irradiated at -78 degrees C in a calibrated Gammacell 60Co irradiator. A three-step protein extraction procedure provided protein samples for the determination of the target size (TS) of the covalently labelled estrogen receptor (ER). From the TS it is shown that ER bound to an antiestrogen was, in whole cells, part of a 265 kDa polypeptide structure if measured in MCF-7 cells at subconfluency, or of a 360 kDa species in superconfluent cells.

Effects of ionizing radiations on proteins. Evidence of non-random fragmentations and a caution in the use of the method for determination of molecular mass.

We have reinvestigated the use of ionizing radiations to measure the molecular mass of water-soluble or membrane proteins. The test was performed by using the most straightforward aspect of the technique, which consists of SDS/PAGE analysis of the protein-fragmentation process. We found that exposure of purified standard proteins to increasing doses of ionizing radiation causes progressive fragmentation of the native protein into defined peptide patterns. The coloured band corresponding to the intact protein was measured on the SDS gel as a function of dose to determine the dose (D37.t) corresponding to 37% of the initial amount of unfragmented protein deposited on the gel. This led to a calibration curve between 1/D37.t and the known molecular mass of the standard proteins whose best fit gave Mr = 1.77 x 10(6)/D37.t at -78 degrees C, i.e. 35% higher than the generally accepted value at that temperature obtained from inactivation studies. However, we have to conclude that this method is useless to determine the state of aggregation of a protein, since, for all the oligomers tested, the best fit was obtained by using the protomeric molecular mass, suggesting that there is no energy transfer between promoters. Furthermore, SDS greatly increases the fragmentation rate of proteins, which suggests additional calibration problems for membrane proteins in detergent or in the lipid bilayer. But the main drawback of the technique arises from our observation that some proteins behaved anomalously, leading to very large errors in the apparent target size as compared with true molecular mass (up to 100%). It is thus unreliable to apply the radiation method for absolute molecular-mass determination. We then focused on the novel finding that discrete fragmentation of proteins occurs at preferential sites, and this was studied in more detail with aspartate transcarbamylase. N-Terminal sequencing of several radiolysis fragments of the catalytic chain of the enzyme revealed that breaks along the polypeptide chains are localized close to the C-terminal end. Examination of the three-dimensional structure of aspartate transcarbamylase suggests that radiolysis sites (fragile bonds) might be localized in connecting loops.