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Scalable methods of recombinant adeno-associated virus (rAAV) production have gained much recent interest as the field of rAAV-mediated gene therapy approaches the clinic. In particular, the production of rAAV vectors in insect cells via the use of recombinant baculovirus technology has proven to be an efficient and scalable means of rAAV production. Here, we describe a method for the production of rAAV serotypes 1 and 2 in insect cells using a simplified baculovirus-AAV expression vector system coupled with particle purification via affinity chromatography. The number of separate baculovirus constructs required for rAAV production was reduced by genetically modifying the AAV rep gene to allow expression of the AAV-encoded replication enzymes, Rep78 and Rep52, from a single mRNA species and combining the modified rep gene with an AAV cap gene expression cassette in a single baculovirus construct. Additionally, we describe lysis, binding, and elution conditions compatible with a commercially available affinity medium (AVB Sepharose High Performance) used to purify rAAV particles to near homogeneity in a single chromatography step. Using the described method, we obtained an average yield of 7 x 10(4) purified rAAV particles per cell (range: 3.7 x 10(4) to 9.6 x 10(4)) from suspension cultures of recombinant baculovirus-infected insect cells.

Cytoplasmic dynein and dynactin interact to drive microtubule-based transport in the cell. The p150Glued subunit of dynactin binds to dynein, and directly to microtubules. We have identified alternatively spliced isoforms of p150Glued that are expressed in a tissue-specific manner and which differ significantly in their affinity for microtubules. Live cell assays indicate that these alternatively spliced isoforms also differ significantly in their microtubule plus end-tracking activity, suggesting a mechanism by which the cell may regulate the dynamic localization of dynactin. To test the function of the microtubule-binding domain of p150Glued, we used RNAi to deplete the endogenous polypeptide from HeLa cells, followed by rescue with constructs encoding either the full-length polypeptide or an isoform lacking the microtubule-binding domain. Both constructs fully rescued defects in Golgi morphology induced by depletion of p150Glued, indicating that an independent microtubule-binding site in dynactin may not be required for dynactin-mediated trafficking in some mammalian cell types. In neurons, however, a mutation within the microtubule-binding domain of p150Glued results in motor neuron disease; here we investigate the effects of four other mutations in highly conserved domains of the polypeptide (M571T, R785W, R1101K, and T1249I) associated in genetic studies with Amyotrophic Lateral Sclerosis. Both biochemical and cellular assays reveal that these amino acid substitutions do not result in functional differences, suggesting that these sequence changes are either allelic variants or contributory risk factors rather than causative for motor neuron disease. Together, these studies provide further insight into the regulation of dynein-dynactin function in the cell.

During directed cell migration, the movement of the nucleus is coupled to the forward progression of the cell. The microtubule motor cytoplasmic dynein is required for both cell polarization and cell motility. Here, we investigate the mechanism by which dynein contributes to directed migration. Knockdown of dynein slows protrusion of the leading edge and causes defects in nuclear movements. The velocity of nuclear migration was decreased in dynein knockdown cells, and nuclei were mislocalized to the rear of motile cells. In control cells, we observed that wounding the monolayer stimulated a dramatic induction of nuclear rotations at the wound edge, reaching velocities up to 8.5 degrees/minute. These nuclear rotations were significantly inhibited in dynein knockdown cells. Surprisingly, centrosomes do not rotate in concert with the nucleus; instead, the centrosome remains stably positioned between the nucleus and the leading edge. Together, these results suggest that dynein contributes to migration in two ways: (1) maintaining centrosome centrality by tethering microtubule plus ends at the cortex; and (2) maintaining nuclear centrality by asserting force directly on the nucleus.

Depression, obesity, diabetes mellitus, and the metabolic syndrome are conditions commonly treated in primary care. The prevalence of each condition separately does not explain the frequency of their co-occurrence. Depression may lead to or exacerbate these endocrine and metabolic conditions. Conversely, these medical conditions may lead to or exacerbate depression. Psychotropic drugs that treat depression may increase appetite with resultant weight gain. Rarely, such agents may be associated with weight loss. We review the potential for psychotropic drugs to alter body weight and provide a table as a guide to drug selection. Unless circumstances dictate otherwise, clinicians should select psychotropic drugs least likely to induce weight gain when treating depressed patients with obesity, diabetes mellitus, or the metabolic syndrome. Even drugs generally thought to be "weight neutral" may occasionally be associated with weight gain. Thus, alerting patients to this potential and due diligence form the cornerstone of weight management in the depressed patient.

Background: The versatility of recombinant adeno-associated vector (rAAV) as a gene delivery system is due to the vector's ability to transduce different cell types as well as dividing and non-dividing cells. Large-scale production of rAAV remains one of the major challenges for continued development of pre-clinical and clinical studies, and for its potential commercialization. The baculovirus expression vectors (BEVS) and insect cells represent a potential method to produce rAAV economically at large scale. This technology uses three different BEVs (Bac-Rep, Bac-GFP, and Bac-VP) each at a multiplicity of infection (MOI) of 3. We reported previously the production of rAAV at 40 L scale using a stirred-tank bioreactor (STB). However, production in larger volumes is limited by the stability of the BEVs and amount of BEVs needed to achieve the target MOI of 3 per BEV. Here, the production parameters were optimized and the baculovirus stability was determined., Methods: The stability of the three types of baculovirus used to produce rAAV was determined for six expansion passages by protein expression analysis. To economize baculovirus, MOI and cell density at time of infection (TOI) were evaluated initially at small scale and then applied to the 10 L scale., Results: An MOI = 0.03 and TOI cell density of 1 x 10(6) cells/mL produced high titer rAAV without comprising yield. To confirm the scalability of the process, rAAV was produced in a 10 L STB using the optimized parameters obtaining a 10x increase in yield ( approximately 1 x 10(14) rAAV DNAse-resistant particles per liter)., Conclusion: These findings contribute to the process development for large-scale production of rAAV for gene therapy applications and its commercialization., (2007 John Wiley & Sons, Ltd)

Background: Peptide YY (PYY) is released from the distal small intestine and colon after meals and reduces appetite by increasing satiety. The amount of PYY released is proportional to calories ingested. Fat ingestion has also been reported to stimulate PYY release., Objective: The objective of the study was to determine whether macronutrient composition influences postprandial serum PYY levels by comparing 1 wk of a weight-maintenance low-carbohydrate, high-fat (LCHF) diet with a low-fat, high-carbohydrate (LFHC) diet., Methods: In this randomized crossover study, 18 obese subjects (14 females, 4 males, mean body mass index 35.6 +/- 2.9 kg/m(2)) were randomly assigned initially to 1 wk of a weight-maintenance LCHF or LFHC diet, after which a test meal of identical composition was given and serum PYY levels were assessed for 2.5 h postprandially. After a 1-wk washout period, subjects were crossed over and retested., Results: After 1 wk, mean postprandial area under the curve PYY after the LCHF test meal was 1.5-fold greater than after the LFHC test meal (P < 0.001). The LCHF diet led to 55% higher levels of postprandial serum PYY levels, compared with the LFHC diet (P = 0.005)., Conclusions: These data show that a LCHF diet stimulates PYY secretion more than a LFHC diet in obese individuals.

Gap junction formation depends on the proper transport of connexin hemichannels to sites of cell-cell contact. Recently in Cell, Shaw et al. implicate microtubule tip tracking proteins in the trafficking of connexin43 to adherens junctions (Shaw et al., 2007). This finding suggests a mechanism for targeted delivery of membrane proteins by microtubule capture at the cortex.

The microtubule motor protein cytoplasmic dynein and its activator dynactin are essential in higher eukaryotes, due to critical roles in vesicular transport and cell division. Neurons are uniquely sensitive to defects in dynein/dynactin function, which affect retrograde axonal transport, neurotrophic factor signaling, neurofilament transport, mRNA localization, neuronal migration, and protein recycling and degradation. Mutations in either dynein or dynactin lead to motor neuron degeneration and loss. Recent progress in understanding the cellular mechanisms of dynein/dynactin function, and the effects of dynein/dynactin dysfunction has provided new insight into the roles of microtubule-based motility in the neuron.

Objectives: As part of a project to map the literature of nursing, sponsored by the Nursing and Allied Health Resources Section of the Medical Library Association, this study identifies core journals cited by general or "popular" US nursing journals and the indexing services that cover the cited journals., Methods: Three journals were selected for analysis: American Journal of Nursing, Nursing 96-98, and RN. The source journals were subjected to a citation analysis of articles from 1996 to 1998, followed by an analysis of database access to the most frequently cited journal titles., Results: Cited formats included journals (63.7%), books (26.6%), government documents (3.0%), Internet (0.5%), and miscellaneous (6.2%). Cited references were relatively current; most (86.6%) were published in the current decade. One-third of the citations were found in a core of 24 journal titles; one-third were dispersed among a middle zone of 94 titles; and the remaining third were scattered in a larger zone of 694 titles. Indexing coverage for the core titles was most comprehensive in PubMed/MEDLINE, followed by CINAHL and Science Citation Index., Conclusions: Results support the popular (not scholarly) nature of these titles. While not a good source for original research, they fulfill a key role of disseminating nursing knowledge with their relevantly current citations to a broad variety of sources.

Malnutrition in advanced cirrhosis may worsen liver function and increase susceptibility to infections. Immune-enhancing nutrition supplements (IENS) may be of value, but their safety in patients with decompensated cirrhosis and history of encephalopathy is unknown. We assessed the safety of Impact Recover (Novartis, St. Louis Park, MN), an orally palatable IENS, in 12 men with hepatic cirrhosis of Child-Turcotte-Pugh (CTP) class B or C, ages 40-60. On day 0, patients were evaluated serially for 6 hours after ingestion of 2 packets of Impact Recover. Despite a transient doubling of the blood ammonia, no cognitive abnormalities were noted on clinical assessment or psychometric testing. Subsequently, patients were instructed to ingest 3 packets per day of Impact Recover for 56 days, after which supplements were stopped. Patients were evaluated in a fasting state on days 0 (baseline), 56 (end of treatment), and 112 (follow-up). One patient was transplanted on day 21, and another died after an urgent cholecystectomy on day 30. The remaining 10 patients completed the study. Mean value of CTP score was 9 (range, 7-11) and mean value of model for end-stage liver disease (MELD) score was 14 (7-21), and there was no change after 8 weeks of IENS. Only 1 experienced transient worsening of encephalopathy after omitting lactulose. Performances on psychometric tests did not change. Transferrin levels increased rapidly with IENS, then returned toward baseline after IENS was stopped. Fasting insulin and peptide YY (PYY) levels also increased, but fasting glucose and hemoglobin A1C did not change. Trends in other nutrition and immune parameters did not reach significance. We conclude that acute and chronic administration of Impact Recover was well tolerated in cirrhotic patients with controlled encephalopathy. Further studies are justified to assess potential efficacy of long-term IENS in preventing infection and slowing progression in advanced cirrhosis.

The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. Dynactin is ubiquitously expressed in eukaryotes, but a G59S mutation in the p150Glued subunit of dynactin results in the specific degeneration of motor neurons. This mutation in the conserved cytoskeleton-associated protein, glycine-rich (CAP-Gly) domain lowers the affinity of p150Glued for microtubules and EB1. Cell lines from patients are morphologically normal but show delayed recovery after nocodazole treatment, consistent with a subtle disruption of dynein/dynactin function. The G59S mutation disrupts the folding of the CAP-Gly domain, resulting in aggregation of the p150Glued protein both in vitro and in vivo, which is accompanied by an increase in cell death in a motor neuron cell line. Overexpression of the chaperone Hsp70 inhibits aggregate formation and prevents cell death. These data support a model in which a point mutation in p150Glued causes both loss of dynein/dynactin function and gain of toxic function, which together lead to motor neuron cell death.

Prolonged infusions of lipid and heparin that achieve high physiological free fatty acid (FFA) concentrations inhibit hepatic (and peripheral) insulin sensitivity in humans. These infusions are composed largely of polyunsaturated fatty acids (PUFA; linoleic and linolenic). It is not known whether fatty acid composition per se affects hepatic glucose metabolism in humans. To address this issue, we examined the impact of enteral infusions of either palm oil (48% palmitic, 35% oleic, and 8% linoleic acids) or safflower oil (6% palmitic, 12% oleic, 74% linoleic acids) in 14 obese nondiabetic subjects. (2)H(2)O was administered to determine the contribution of gluconeogenesis to endogenous glucose production (EGP), and a primed continuous infusion of [6,6-(2)H]glucose was administered to assess glucose appearance. As a result of the lipid infusions, plasma FFA concentrations increased significantly in both the palm oil (507.5 +/- 47.4 to 939.3 +/- 61.3 micromol/l, P < 0.01) and safflower oil (588.2.0 +/- 43.0 to 857.8 +/- 68.7 micromol/l, P < 0.01) groups after 4 h. EGP was similar at baseline (12.4 +/- 1.8 vs. 11.2 +/- 1.0 micromol x kg FFM(-1) x min(-1)). During a somatostatin-insulin clamp, the glucose infusion rate was significantly lower (AUC glucose infusion rate 195.8 +/- 50.7 vs. 377.8 +/- 38.0 micromol/kg FFM, P < 0.01), and rates of EGP were significantly higher (10.7 +/- 1.4 vs. 6.5 +/- 1.5 micromol x kg FFM(-1) x min(-1), P < 0.01) after palm oil compared with safflower oil, respectively. Baseline rates of gluconeogenesis and glycogenolysis were also similar. However, after lipid infusion, rates of glycogenolysis were suppressed by safflower oil but not by palm oil. Thus these studies demonstrate, for the first time in humans, a differential effect of saturated fatty acids and PUFA on hepatic glucose metabolism.

Glucose administration to rodents acutely stimulates leptin secretion. To investigate the mechanism, rats were infused intravenously with various concentrations of glucose, and plasma leptin concentrations were measured with time. The osmolality of the infusates was equalized with various concentrations of carbohydrates that are not metabolized. Hyperosmolar glucose stimulates leptin secretion in a dose-dependent manner, with peak plasma leptin concentrations occurring approximately 3 h after the end of the glucose infusion. Hypertonic infusions of galactose, mannitol, and sodium chloride independently stimulate leptin secretion with approximately one-half the strength of equivalent osmolar concentrations of glucose. Peak plasma leptin concentrations occur approximately 4 h after the end of the hypertonic solution infusion. Hypertonic solutions of mannitol do not stimulate leptin secretion in vasopressin-deficient or in adrenalectomized animals. In conclusion, intravenous infusions of hypertonic glucose and hypertonic mannitol independently stimulate leptin secretion. Hyperosmolality stimulates leptin secretion by a vasopressin-adrenal mechanism.

Dietary fatty acid composition modifies hepatic lipid metabolism. To determine the effects of fatty acids on hepatic triglyceride storage, rats were fed diets enriched in carbohydrates (control), fish oil, or lard. After 4 weeks, the animals were fasted overnight. In the morning, the animals were either sacrificed or fed 8 g of their respective diets before sacrifice. Animals ingested more food calories with diets containing fish oil than with other diets. However, fish oil-fed animals weighed less and had less body fat. In fish oil-fed animals, liver triglyceride was lower by 27% (P <.05) and 73% (P <.01) than in control- and lard-fed animals, respectively. Fish oil altered the postprandial gene expression of hepatic regulators of fatty acid degradation and synthesis. Fish oil feeding blunted the normal postprandial decline in fatty acid degradation genes (PPARalpha, CPT1, and ACO) and blunted the normal postprandial rise in triglyceride synthesis genes (SREBP1-c, FAS, SCD-1). Therefore, the direct postprandial effect of fish oil ingestion decreases the propensity for hepatic triglyceride storage. In conclusion, n-3 polyunsaturated fatty acids decrease total body weight, total body fat, and hepatic steatosis.

Background: Autonomic dysreflexia (AD) is a frequent, serious acute syndrome that occurs in patients with spinal cord lesions at level T6 and above. The syndrome is caused by massive sympathetic discharge that is triggered by a noxious stimulus below the level of the spinal cord lesion. Pheochromocytomas are rare tumors that present with symptoms similar to AD., Methods: Case Report., Findings: A 50-year-old man with C7 American Spinal Injury Association scale A tetraplegia presented with episodes of severe headaches and paroxysmal hypertension. He was diagnosed with AD. Despite resolving bladder and bowel problems, he continued to have hypertensive episodes. A CT scan of the abdomen revealed a heterogeneous left adrenal mass. Further workup revealed significantly elevated serum and 24-hour urinary catecholamines. Clonidine failed to fully suppress the markedly elevated concentrations of serum catecholamines. These biochemical findings were consistent with the diagnosis of pheochromocytoma. Prior to surgery, the patient was treated with alpha-receptor blockers and volume expansion with intravenous fluids. A left adrenalectomy was performed. The surgical specimen revealed that the adrenal gland was expanded by a spherical mass. The pathologic report was benign pheochromocytoma of the left adrenal gland., Conclusion: Clinical symptoms and hypertensive episodes resolved following adrenalectomy. To our knowledge, this is the first reported case of a pheochromocytoma in an individual with spinal cord injury.

The interrelationship between insulin and leptin resistance in young Fischer 344 (F344) rats was studied. Young F344 and Sprague-Dawley (SD) rats were fed regular chow. F344 animals had two- to threefold higher insulin and triglyceride concentrations and increased stores of triglycerides within liver and muscle. F344 animals gained more body fat. Both acyl-CoA oxidase (ACO) and carnitine palmitoyltransferase I gene expression were 20-50% less in F344 animals than in age-matched SD animals. Peroxisome proliferator-activated receptor-alpha gene expression was reduced in 70-day-old F344 animals. Finally, resistin gene expression was similar in 70-day-old SD and F344 animals. Resistin gene expression increased fivefold in F344 animals and twofold in SD animals from 70 to 130 days, without a change in insulin sensitivity. We conclude that young F344 animals have both insulin and leptin resistance, which may lead to diminished fatty oxidation and accumulation of triglycerides in insulin-sensitive target tissues. We did not detect a role for resistin in the etiology of insulin resistance in F344 animals.

Leptin is a hormone that is secreted by fat cells and has roles in body weight regulation, glucose metabolism, reproduction, and other neuroendocrine functions. The purpose of this study was to determine whether the secretagogues, insulin, glucose, and pyruvate, enhance leptin secretion by increasing leptin synthesis, or whether these secretagogues stimulate the quantal release of a stored cytosolic pool of leptin. We found that in the absence of secretagogues, the rate of leptin secretion from isolated rat adipocytes approximately equals the rate of leptin synthesis. For 60 min after the addition of secretagogues, leptin synthesis rapidly increases, with little or no leptin secretion; leptin increases intracellularly by approximately 60% (P < 0.05). After 60 min, leptin is significantly released from cells. At 120 and 240 min, secretagogues enhance leptin secretion into the medium by 35% (P < 0.05) and 40% (P < 0.01), respectively. Cycloheximide prevents the synthesis and the secretagogue-mediated secretion of leptin. Monensin, an inhibitor of protein translocation, has no effect on leptin synthesis, but it blocks the secretagogue-mediated secretion of leptin. These findings suggest that secretagogues enhance leptin release by increasing leptin synthesis, rather than by enhancing the release of a preexisting cytosolic pool of leptin.

The prevalence, risk factors, and prevention opportunities of hepatitis C virus (HCV) infection were studied in a large sample of 698 young adult injection drug users (IDUs) in Chicago, 18-30 years old. Participants were recruited between 1997 and 1999 by using street outreach, targeted advertising, and chain-referral methods. HCV infection prevalence was 27% and was strongly associated with both age and duration of injecting (P<.001). In multivariable analysis, sexual behaviors were unrelated to seropositivity. Independent drug-related risk factors included frequent injection, heavy crack smoking, injecting in a shooting gallery, and syringe-mediated sharing. Urban residents were more likely than suburban residents to be infected. Most research on hepatitis C has shown rapid spread of infection among IDUs, but these findings underscore that opportunities to identify IDUs uninfected with HCV may be greater than assumed and emphasize the need to target younger, newer IDUs.

The secretion of leptin is dually regulated. In fasting animals, plasma leptin concentrations reflect body fat stores, whereas the incremental leptin response to fasting or refeeding most likely reflects insulin-mediated energy flux and metabolism within adipocytes. Impaired secretion of leptin in either pathway could result in obesity. We therefore measured plasma leptin concentrations in fasted animals and plasma leptin concentrations after an intravenous glucose infusion in a rat model of obesity. Young Sprague-Dawley (S-D) and Fischer 344 (F344) rats had similar percent body fat and fasting glucose and fasting leptin concentrations. However, F344 animals had higher insulin concentrations and leptin responses to intravenous glucose than did the S-D animals. The animals were then fed a control or high-fat diet for 6 wk. High-fat fed animals gained more weight and body fat than did the control fed animals. Control and high-fat fed F344 animals gained approximately 40% (P < 0.0001) more weight and >100% (P < 0.01) more body fat than did the S-D animals. Fasting leptin concentrations and leptin concentrations after intravenous glucose infusions and feeding were more than double (P < 0.05) in F344 animals compared with S-D animals. Whether an animal is fed a control or high-fat diet had little effect on the leptin response to intravenous glucose. In conclusion, young, lean F344 animals, before the onset of obesity, demonstrated a greater acute leptin response to intravenous glucose than similarly lean S-D animals. After a 6-wk diet, F344 animals had a greater percent increase in body weight and insulin resistance and exhibited higher fasting leptin concentrations and a greater absolute leptin response to intravenous glucose compared with the S-D animals. The chronic diet (control or high fat) had little impact on the acute leptin response to intravenous glucose. F344 animals exhibit leptin resistance in young, lean animals and after aging and fat accumulation.

Rodent leptin is secreted by adipocytes and acutely regulates appetite and chronically regulates body weight. Mechanisms for leptin secretion in cultured adipocytes were investigated. Acutely, energy-producing substrates stimulated leptin secretion about twofold. Biologically inert carbohydrates failed to stimulate leptin secretion, and depletion of intracellular energy inhibited leptin release. There appears to be a correlation between intracellular ATP concentration and the rate of leptin secretion. Insulin increased leptin secretion by an additional 25%. Acute leptin secretion is calcium dependent. When incubated in the absence of calcium or in the presence of intracellular calcium chelators, glucose plus insulin failed to stimulate leptin secretion. In contrast, basal leptin secretion is secreted spontaneously and is calcium independent. Adipocytes from fatter animals secrete more leptin, even in the absence of calcium, compared with cells from thinner animals. Acute stimulus-secretion coupling mechanisms were then investigated. The potassium channel activator diazoxide and the nonspecific calcium channel blockers nickel and cadmium inhibited acute leptin secretion. These studies demonstrate that intracellular energy production is important for acute leptin secretion and that potassium and calcium flux may play roles in coupling intracellular energy production to leptin secretion.

Background: The hormone leptin has putative roles in both body weight homeostasis (chronic) and satiety (acute). To determine if this dual regulation is observed in hospitalized, undernourished patients, serum leptin concentration was measured before and during total parenteral nutrition (TPN) infusion., Methods: Six consecutive patients were considered undernourished, as assessed by an independent multidisciplinary nutrition team, and TPN was prescribed at an initial rate of between 5023.2 and 7333.2 kJ in the first 24 hours. Serum leptin, insulin, and glucose were measured before the infusion and at 3 and 22 hours after initiation of TPN., Results: Baseline serum leptin concentrations correlated well with the patient's body mass index (BMI; r2 = .85, p<.05). Three hours of TPN infusion produced only modest changes in circulating leptin. However, after 22 hours, leptin concentrations increased by 1.8+/-0.5-fold (p<.05), and this increase was independent of any change in body weight., Conclusions: Basal leptin concentrations correlate well with BMI. TPN induces a rise in leptin concentration independent of body weight. Leptin secretion is dually regulated in hospitalized, undernourished patients.

Purpose: This paper reports on the development of a new phantom for spiral CT. The phantom meets the increased demands on phantom z-axis uniformity in order that objects from the CT slice, immediately above and below the CT slice of interest, do not contribute perturbing information to the reconstructed CT slice., Material and Methods: The phantom depends on formulation of tissue-like materials that can be cast and produced in both geometric and anthropomorphic shapes with sufficient z-axis length to enable unperturbed CT slices of test objects of interest. These materials are then used to produce a series of test objects of CT image quality including low contrast samples that do not require volume averaging or mixing of solutions, and that can reflect sub-slice thickness test objects and supra-slice thickness test objects., Results: The overall phantom and its individual test objects provides meaningful tests of spiral CT image quality including slice sensitivity, CT number linearity and tests of high and low contrast resolution. Schematic designs and actual CT scans are shown., Conclusion: The new spiral phantom appears to meet the increased demands of spiral CT on phantom design, particularly z-axis length, and requirements for low contrast resolution test objects.

In the rat, the ob gene product, leptin, putatively regulates energy balance via appetite control and energy expenditure. Bile acids in the intestinal lumen are necessary for efficient absorption of dietary lipids and may trigger the release of regulatory peptides. To investigate whether bile acids play a role in leptin gene expression, we altered the bile acid pool and then measured leptin mRNA levels in adipose tissue. Rats fed cholic acid (1% of chow wt/wt) for 2 weeks did not gain weight as rapidly as pair-fed control animals. Despite the lower weight, normalized leptin mRNA levels were 24% greater in cholic acid-fed rats compared with controls. Conversely, cholestyramine, a bile acid sequestrant, in chow (5% wt/wt) resulted in a 26% decline in leptin mRNA. Ligation of the common bile duct or chronic biliary diversion, experimental manipulations that decreased the intestinal concentration of bile salts, decreased leptin gene expression by 30% and 50%, respectively. A fluid and electrolyte (F/E) solution with and without taurocholate (36 micromol/h x 100 g rat[-1]) was then infused for 12 hours into the duodenum in animals with chronic biliary diversion. Taurocholate infusion resulted in a fourfold increase in steady-state adipocyte leptin mRNA levels compared with F/E infusion. Intravenous infusion of taurocholate or incubation of cultured adipocytes with taurocholate had no effect on leptin mRNA levels. We conclude that bile acids upregulate leptin gene expression indirectly, probably via effects on the absorption of dietary lipids or the release of neurohumoral mediators.

Leptin, a putative satiety hormone in rodents, is acutely regulated by fasting and refeeding. To determine the role of satiety hormones that are secreted by the gastrointestinal tract on leptin regulation, leptin mRNA and serum concentrations were measured after feeding rats similar calories with standard chow or infusion of total parenteral nutrition into the duodenum or intravenously. We have demonstrated that leptin gene expression and hormone secretion into the circulation are stimulated equally in the three experimental paradigms; it is unlikely that satiety factors secreted by the intestinal tract play a significant role in leptin regulation. Furthermore, intravenous infusion of individual components of TPN demonstrated that intravenous glucose infusion was mostly responsible for stimulation of the leptin gene and hormone secretion.

Introduction: Biofeedback training is an effective modality for the treatment of chronic constipation and fecal incontinence. In general, patients express satisfaction and perceive functional improvement following biofeedback therapy; however, quantifying these observations has been difficult., Aim: This study was undertaken to evaluate the physiologic benefits of biofeedback therapy as reflected by noninvasive electromyography parameters., Methods: Fifty-five patients who underwent computerized electromyography-based biofeedback treatment at our institution between July 1993 and July 1995 were identified. Noninvasive electromyographic testing was performed before, during (weekly), and at completion of training. Mean number of weekly sessions was seven (range, 5-11). Short-term and ten-second contractions (amplitude/microV), sustained contractions (endurance, in seconds), and net strength (microV) of the external anal sphincter before and after biofeedback were compared for differences., Results: There were 30 patients with chronic constipation, mean age, 65.3 (range, 33-86) years, composed of 24 women, and 25 patients with fecal incontinence, mean age 66 (range, 34-85) years, composed of 12 males. Statistically significant improvement in endurance and net strength following biofeedback training was noted in both the constipated and the fecal incontinence groups. Fifty-three of 55 (96.4 percent) patients expressed 50 to 100 percent subjective satisfaction after biofeedback therapy. Forty-six of 55 (83.6 percent) patients demonstrated individually improved endurance., Conclusions: Sphincter endurance and net strength, as measured by noninvasive electromyography, significantly improve following biofeedback therapy in both constipated and fecal incontinence patients. These data suggest that endurance and net strength may be useful tools in assessing a benefit from biofeedback training in these patients.

Purpose: Biofeedback treatment is often offered to patients in colorectal centers; however, standards of treatment are still lacking. A dedicated team approach is desirable but difficult to coordinate. We present our three-year experience of electromyographic-based biofeedback treatment offered within a multicenter, statewide organization., Methods: Between October 1992 and October 1995, 188 patients completed a biofeedback treatment program in one of five coordinated centers within a 200-mile radius. A unified common database was established and continuously updated. A colorectal surgeon served as statewide director, and dedicated teams were established at each location. Each local team included the medical director and a certified biofeedback therapist and had access to a dietitian and a nurse data coordinator. Electromyographic-based biofeedback sessions were given weekly, and a home trainer program was established., Results: A total of 116 patients with chronic constipation had a mean of eight (range, 2-14) weekly sessions. A total of 72 patients with fecal incontinence had a mean of seven (range, 2-11) weekly sessions. A total of 84 percent of the constipated and 85 percent of the incontinent patients had significant improvement with biofeedback treatment. Patient compliance and satisfaction were high. Constipated patients increased the mean number of weekly unassisted bowel movements from 0.8 to 6.5. Incontinent patients decreased the mean number of weekly gross incontinence episodes from 11.8 to 2., Conclusions: Biofeedback treatment can be extremely successful in both incontinent and constipated patients. A large geographic area can be covered with coordinated centers in which each dedicated team uses a unified treatment protocol, and a common database is established.

The phenotype and allelic expression of the insulin receptor gene is presented in a family with a patient with type A insulin resistance. Compared to controls, insulin receptor binding in transformed lymphocytes was 100%. 33% and 13% in the father, mother and proband, respectively. Reduced insulin receptor binding co-segregated with altered insulin receptor mRNA expression; the mother and daughter expressed eight insulin receptor mRNA species, including a set of four normal sized and a set of four shorter mRNA transcripts. In the proband the levels of the normal sized mRNA transcripts were suppressed relative to the shorter transcripts. Reverse polymerase chain reaction (PCR) revealed that the shorter transcripts contained an in-frame deletion of exon 2. Sequencing of the entire insulin receptor coding region revealed a paternally inherited A to T substitution in nucleotide 3205, converting isoleucine 996 to phenylalanine, which does not co-segregate with reduced binding. Therefore, we hypothesize that two findings are necessary for the presentation of type A insulin resistance in this patient: an in-frame deletion of the insulin receptor exon 2 that codes for amino acids crucial for insulin binding: and an inhibition of expression of the paternal insulin receptor allele.

We present 5.1 kb of the 3' noncoding region sequence of the human insulin receptor gene and identification of four functional polyadenylation domains responsible for 3'-end processing of the 5.4, 6.9, 8.0 and 9.4 kb human insulin receptor mRNA, respectively. The insulin receptor gene contains five putative polyadenylation sites (P1-P5), located 5160, 6502, 7488, 8945 and 8957 base pairs (bp) downstream from the translational initiation site. All putative polyadenylation sites are flanked by upstream AU rich and downstream GU rich regions which regulate mRNA stability and mRNA cleavage, respectively. Also, two RNA stem-loop structures have been identified. To determine its role on gene expression, a reporter gene was constructed containing various lengths of the insulin receptor 3' UTR and transiently transfected into COS 7 cells. A 539 bp fragment (4897-5436 bp downstream from the IR translational initiation site) inhibited CAT expression by 5-6 fold. Further downstream addition of 1169 bp of the insulin receptor 3' untranslated region enhanced gene expression by 2-fold. These studies provide evidence that the insulin receptor 3' untranslated region can modulate gene expression.

Exposure to naturally occurring estrogens during critical periods of development can alter morphologic and physiologic markers of sexual differentiation. The current experiment characterizes the effects of in utero treatment with genistein, an isoflavonoid phytoestrogen, on birth weight, anogenital distance (AGD) at birth. GnRH stimulated luteinizing hormone (LH) secretion, volume of the sexually dimorphic nucleus in the preoptic area of the hypothalamus (SDN-POA), puberty onset, and vaginal cyclicity. Pregnant Charles River CD rats were injected sc daily on gestation day 16-20 with either 25,000 micrograms genistein (G25), 5,000 micrograms genistein (G5), 5 micrograms diethylstillbestrol (DES), 50 micrograms estradiol benzoate (E), or corn oil alone for controls. Birth weights and anogenital distance was taken and exposed progeny were subsequently used in two experiments. In Experiment 1 intra-atrial catheters were placed in adult castrated rats, GnRH was given iv, serial blood samples were drawn and sera were assayed for LH by radioimmunoassay (RIA). Brains obtained by subsequent decapitation were saved for histology. In Experiment 2, females were monitored for timing of vaginal opening as a marker of puberty onset, and vaginal smears were taken to monitor cyclicity. G25-treated females and DES- and E-treated animals of both sexes had decreased weights at birth compared with controls. G5- and E-treated animals of both sexes and DES males had smaller AGD than controls. No significant differences in pituitary responsiveness to GnRH were found among treatment groups. There was a nonsignificant decrease in SDN-POA volume in G5-treated females while DES- and E-treated females had increased SDN-POA volume compared with controls. G5-treated females had delayed puberty onset, and DES-treated females had atypical vaginal cycles in comparison with controls. The results confirm that prenatal exposure to estrogens in the environment can influence sexual differentiation. Our previous experiments have demonstrated that castrate female rats exposed as neonates to genistein have decreased pituitary responsiveness to GnRH challenge and enlarged SDN-POA volume in comparison with controls. Prenatal genistein at these dosages did not significantly alter these markers. However, genistein did mimic other estrogens' effects on AGD and birth weight and had a unique influence on puberty onset. Not only are genistein's effects different from other estrogens, but dosage and timing of exposure during development appear to be important factors in genistein's ability to modify these end points.

To determine the mechanism of glucocorticoid mediated enhancement of insulin receptor (IR) gene expression, we cotransfected a glucocorticoid receptor expression vector and a plasmid containing a reporter gene driven by an MMTV or IR promoter into COS 7 cells. Dexamethasone (Dex) increased MMTV promoter activity by 100% but had no effect on IR promoter activity. In the glucocorticoid responsive breast cancer cell line, MCF-7, Dex increased IR mRNA by 60%, and increased the IR mRNA half-life from approximately 6hrs to > 24 hrs. No glucocorticoid responsive element could be located in the insulin receptor 3' untranslated region. Glucocorticoids stabilize IR mRNA.

We previously demonstrated that activation of the insulin-receptor promoter occurs between Xho I and HindIII restriction enzyme sites located 877 and 578 bp upstream, respectively, from the translational initiation site. Deletion mutants 5' of this promoter region were constructed with the reporter gene, CAT, and transiently transfected into HepG2 and MCF-7 cells. This study demonstrated that most of the promoter activity could be localized to a 40-bp region between -618 and -578. When attached to a heterologous SV40 early promoter, this 40-bp insulin-receptor regulatory region, in either orientation, stimulated the SV40 early promoter by approximately two- to threefold after transfection into HepG2 cells. EMSA demonstrated that the purified transcription factor, Sp1, binds to this transcription activator. DNA binding of protein obtained from crude HepG2 nuclear extracts demonstrated electrophoretically retarded bands that competed for the consensus Sp1 element; these bands were not observed in a similar analysis with crude MCF-7 nuclear extracts. However, in both HepG2 and MCF-7 cells, a protein was identified that specifically binds to this important insulin-receptor promoter region, but does not bind to the Sp1 consensus element. We conclude that activation of insulin-receptor gene transcription occurs in a 40 bp region 578 bp upstream from the translational initiation site, and that Sp1 and another nuclear factor other than Sp1 may be important in regulating transcription in HepG2 cells.

Posttranscriptional mechanisms play important roles in insulin receptor gene regulation; variability in cellular insulin receptor number and the growth arrest-mediated increases in insulin receptor mRNA are secondary to changes in insulin receptor mRNA stability. Therefore, further characterization of the pathways and kinetics of insulin receptor mRNA degradation were investigated. The insulin receptor mRNA in the insulin receptor-rich Hep G2 cells is more stable compared with the insulin receptor-sparse MCF-7 cells. Growth arrest results in a significant rise in insulin receptor mRNA in both cell lines. The increase in mRNA is caused by changes in mRNA stability. The half-life of the insulin receptor mRNA in growth-arrested cells is approximately three times that of proliferating cells. The insulin receptor gene contains four polyadenylation sites that produce four species of mRNA of 5.4, 6.9, 8.0, and 9.4 kilobases (kb). The mRNA species are not coordinately regulated. The ratio of the most abundant species (9.4/6.9) is significantly larger in growth-arrested cells compared with proliferating cells. By utilizing a specific cDNA probe for the 9.4-kb mRNA species, it was determined that the diminished 9.4/6.9 ratio in proliferating cells was caused by a more rapid rate of the 9.4-kb mRNA degradation. The kinetics of insulin receptor mRNA degradation were investigated. Insulin receptor mRNA levels were reduced to 56% of their base line within 6 h when growth-arrested cells were stimulated to proliferate; protein inhibition with cycloheximide completely inhibited the decline in insulin receptor mRNA.

The mechanisms by which glucocorticoids (GC) inhibit some actions of vitamin D [1,25-(OH)2D3] are not well understood, but there is growing evidence that GC alter vitamin D receptor (VDR) number. We studied the effects of dexamethasone (DEX) on VDR number and mRNA in the human osteosarcoma cell line, MG-63. The effects of DEX on 1,25-(OH)2D3 binding were examined by incubating confluent cells overnight in media without or with 10(-6) M DEX. DEX decreased VDR number (B max) by approximately 70% (110 versus 32 fmol/mg cellular protein, p less than 0.001) without significantly changing the apparent affinity (K'D) of 1,25-(OH)2D3 for its receptor (3.8 versus 2.2 x 10(-10) M, p greater than 0.05). Overnight incubation of MG-63 cells with DEX produced a time- and dose-responsive decrease in VDR mRNA compared to untreated controls (p less than 0.01). To determine the mechanism of the DEX-mediated decrease in VDR mRNA, the effect of DEX on VDR mRNA stability was studied. We found that the half-life for the VDR mRNA was approximately 5.7 h and was not significantly changed when the cells were incubated with DEX (approximately 6.3 h). We conclude that DEX decreases both VDR number and mRNA in MG-63 osteosarcoma cells. Since the half-life of VDR mRNA was not significantly modified by dexamethasone, glucocorticoids appear to decrease VDR mRNA by inhibiting VDR gene transcription or by affecting the processing of VDR mRNA.

Numerous physiological agents and conditions modulate cellular insulin sensitivity by downregulating or upregulating total cellular insulin receptors. In this study, we examined the effects of replacing complete medium in the absence or presence of insulin on the regulation of insulin-receptor gene expression in cultured human hepatoma cells (HepG2). Failure to replace complete medium resulted in growth arrest of HepG2 cells and a six- to sevenfold increase in insulin-receptor mRNA due to the prolongation of insulin-receptor mRNA half-life. Northern analysis revealed multiple insulin-receptor mRNA species; the largest species (11 kilobases) was disproportionately increased in growth-arrested cells. High concentrations of insulin (500 ng/ml) induced a 33.8% decrease in the abundance of insulin-receptor mRNA (n = 14). At lower concentrations, a trend of inhibition was observed but was not statistically significant. Insulin (500 ng/ml) did not affect insulin-receptor mRNA stability. The effect of conditioned media, insulin, and dexamethasone on insulin-receptor promoter activity was also examined. Various constructs of the 5'-flanking region of the insulin-receptor gene were attached immediately upstream to a chloramphenicol acetyltransferase (CAT) reporter gene and transiently transfected into HepG2 cells via a pBR322-derived plasmid (pCAT). In cells replaced with complete medium, 12 and 118% of the promoter activity was contained within 578 and 877 base pairs, respectively, from the major translational initiation site. Conditioned media from growth-arrested cells in culture for 7 days increased promoter activity approximately twofold in 48 h. However, this increase failed to localize to any specific region on the insulin-receptor promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

The delivery of insulin and the insulin receptor into an intracellular compartment may be important for eliciting some of the biologic responses of the cell to the hormone. Internalization of insulin-receptor complexes in cells from hyperinsulinemic type II diabetic patients is diminished, suggesting a possible role for this cellular process in insulin resistance. To examine whether hyperinsulinemia contributes to defective insulin-receptor processing in vitro, cultured hepatoma cells (HepG2) were incubated with high concentrations of (500 ng/ml) insulin from 1-3 days. Insulin induced a decrease in the number of total and surface insulin receptors within 24 hours; however, the hormone did not mediate a change in the number of intracellular receptors. The cellular itinerary of control and down-regulated receptors were then compared. Insulin mediated internalization of down-regulated receptors was impaired compared to control receptors; however, the down-regulated receptors that were internalized recycled back to the plasma membrane more efficiently. By covalently labeling the insulin receptor with the photoactive insulin derivative, 125I-NAPA-DP-insulin, it was demonstrated that the rates of receptor degradation of down-regulated and control receptors were similar. These results suggest that incubating HepG2 cells with high concentrations of insulin alters the cellular itinerary of the insulin receptor.

A figure of '8' magnetic coil (MC) was used to stimulate focally the motor cortex of two adult, traumatic quadriplegics and three normal adults. The two patients were injured approximately 2 years previously and had intense physiotherapy, including biofeedback training of biceps and deltoid muscles, respectively, which were the most caudal muscles spared. The focal MC elicited compound motor action potentials (CMAPs) from these muscles from a much wider area of scalp than in the normal subjects. Latency of biceps and deltoid CMAPs were inversely related to CMAP amplitude. A reorganization of the motor cortical projection system is inferred, in which areas normally eliciting digit movements instead activate muscles in quadriplegics just above the spinal level. The reorganization applies also to the central sense of movement normally elicited by focal frontal cortex stimulation. Possible mechanisms of the reorganization and an implication for rehabilitation are discussed.

The cellular itinerary and processing of insulin and proinsulin were studied to elucidate possible mechanisms for the observed in vivo differences in the biologic half-lives of these two hormones. A rat fibroblast cell line transfected with a normal human insulin receptor gene was used. Due to gene amplification, the cells express large numbers of receptors and are ideal for studying a ligand, such as proinsulin, that has a low affinity for the insulin receptor. Competitive binding at 4 degrees C showed that the concentration of unlabeled insulin and proinsulin that is needed to displace 50% of tracer insulin or proinsulin was 0.85-0.95 nM and 140-150 nM, respectively. Binding to surface receptors and internalization occur at rates that are four to five times faster in cells incubated with insulin compared with proinsulin. Chloroquine led to an increase in cell-associated radioactivity of approximately 1.4-fold in cells incubated with insulin or proinsulin, but inhibited the appearance of degraded insulin by 54% and degraded proinsulin by only 10%. To study the fate of internalized ligand, cells were incubated with insulin and proinsulin until steady state binding occurred. Surface bound ligand was removed by an acid wash and the remaining cell-associated radioactivity represented internalized ligand. Cells were then reincubated in 37 degrees C buffer and the cell-associated radioactivity and radioactivity released into the medium were analyzed by TCA precipitation, Sephadex G-50, and HPLC. The results demonstrated that proinsulin more readily bypasses the intracellular degradative machinery and is therefore released intact from the cell via the retroendocytotic pathway. These results may help to explain the prolonged metabolic clearance rate and biologic responsiveness of proinsulin in vivo.

In an era where funding for social service and related agencies is becoming increasingly scarce, the sharing of staff, resources, and expertise between multiple agencies is one way to compensate and by so doing, improve therapeutic recreation service delivery. This concept, known as networking, is one that was adopted by a group of agencies located in Toledo, Ohio. Through the communication of recreation and recreation related staff, these agencies have joined to provide therapeutic recreation programs and services to children and youth in the area. This article examines the concept of interagency programming and provides a historical discussion of the program's formation, the present services offered, as well as its applicability to other agencies and areas.

Spinal cord monitoring during operation is of increasing importance in the prevention of injury. However, there is no direct monitor of the motor tracts available. We have reported a system using direct stimulation of the area overlying the motor tract between the intermediolateral sulcus and the dentate ligament in cats. This produces a 100-m/second signal with later components, which is abolished by section of the motor area, but not by section of the dorsal columns or the anterior quadrant of the spinal cord. Such stimulation also produces motor movement when the correct frequency is used. We now report the first application of this technique in humans, in whom we found the same 100-m/second signal, as well as slower components. We were able to elicit distal limb motor movement with stimulation of the motor tract area, but not with stimulation of the dorsal column area. This technique can be used either in open surgical cases or percutaneously and should provide an additional valuable technique for assessing spinal cord function.

The cellular processing of insulin and insulin receptors was studied using a rat fibroblast cell line that had been transfected with a normal human insulin receptor gene, expressing approximately 500 times the normal number of native fibroblast insulin receptors. These cells bind and internalize insulin normally. Biochemical assays based on the selective precipitation by polyethylene glycol of intact insulin-receptor complexes but not of free intracellular insulin were developed to study the time course of intracellular insulin-receptor dissociation. Fibroblasts were incubated with radiolabeled insulin at 4 degrees C, and internalization of insulin-receptor complexes was initiated by warming the cells to 37 degrees C. Within 2 min, 90% of the internalized radioactivity was composed of intact insulin-receptor complexes. The total number of complexes reached a maximum by 5 min and decreased rapidly thereafter with a t 1/2 of approximately 10 min. There was a distinct delay in the appearance, rate of rise, and peak of intracellular free and degraded insulin. The dissociation of insulin from internalized insulin-receptor complexes was markedly inhibited by monensin and chloroquine. Furthermore, chloroquine markedly increased the number of cross-linkable intracellular insulin-receptor complexes, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. These findings suggest that acidification of intracellular vesicles is responsible for insulin-receptor dissociation. Physical segregation of dissociated intracellular insulin from its receptor was monitored, based on the ability of dissociated insulin to rebind to receptor upon neutralization of acidic intracellular vesicles with monensin. The results are consistent with the view that segregation of insulin and receptor occurs 5-10 min after initiation of dissociation. These studies demonstrate the intracellular itinerary of insulin-receptor complexes, including internalization, dissociation of insulin from the internalized receptor within an acidified compartment, segregation of insulin from the receptor, and subsequent ligand degradation.

The processing and trafficking of insulin in cultured rat hepatocytes were studied. A time course of binding of radiolabeled insulin to hepatocytes at 37 C revealed a rapid rise in cell-associated radioactivity that reached a steady state by 20 min. Using an acid medium to extract insulin bound to surface receptors, the time courses of receptor binding and internalization of the ligand were characterized. The earliest event in insulin processing was the binding of insulin to surface receptors, reaching steady state by 20 min with a t1/2 of 4 min. The internalization rate of ligand was initially slower than the binding rate, with a t1/2 of 6 min. Similar internalization rates of the insulin receptor were found by measuring the trypsin sensitivity of hepatocyte insulin receptors covalently occupied with a photo-affinity-labeled derivative of insulin [( 125I]B2 (2-nitro-4-azido-phenylacetyl)Des-PheB1-insulin). At steady state, the internalized ligand and receptor comprised approximately 40-45% of the cell-associated radioactivity. The time course of intracellular degradation was assessed by trichloroacetic acid (TCA) precipitability and Sephadex G-50 gel chromatography of solubilized cells containing only internalized radioactivity. Intracellular TCA-soluble and low mol wt degradation products first appeared by 5 min and were released from the cell 3 min later. Chloroquine (100 microM) completely inhibited the formation of intracellular low mol wt degradation products as well as their appearance in the medium. The release of intracellular radioactivity was assessed by first removing surface-bound insulin with acid extraction. Eighty percent of the intracellular radioactivity was released in 45 min with a t1/2 of 8 min. The released radioactivity was assessed by TCA precipitability and gel chromatography. These results demonstrate that after 20 min, 43% of the released intracellular radioactivity is intact insulin. The percentage of intact insulin released increases in a dose-dependent fashion as the amount of insulin bound and internalized increases. In conclusion, the earliest event in insulin processing is binding to surface receptors. After a short delay, insulin and its receptor are internalized and trafficked into either a chloroquine-sensitive degradative pathway or a chloroquine-insensitive retroendocytotic pathway. The amount of insulin that traverses the nondegradative retroendocytotic pathway is proportional to the amount of insulin bound and internalized by the cell.

A variety of ligands internalized by receptor-mediated endocytosis follow a short circuit pathway that does not lead to degradation but results in rapid exocytosis of intact ligand, a process termed retroendocytosis. We studied the time course of [125I]iodoinsulin processing and retroendocytosis after internalization in isolated rat adipocytes. After steady state binding and internalization, surface receptor-bound insulin was removed by exposing cells to a low pH at low temperatures. The cells containing internalized [125I]iodoinsulin were reincubated in fresh medium; subsequently, the radioactivity remaining within the cells and released into the medium were analyzed at various times by trichloroacetic acid (TCA) precipitation, Sephadex G-50 gel filtration, and reverse phase HPLC. Cell-associated radioactivity progressively decreased after reincubation in 37 C buffer, with 50% released in 9 min and 85% by 45 min. In the media, TCA-precipitable material appeared quickly, with a t1/2 of 2 min, and plateaued by 10 min. TCA-soluble material was released continually throughout the 45-min period. The release of both TCA-precipitable and TCA-soluble material was temperature and energy dependent. Sephadex G-50 chromatography demonstrated the loss of insulin from the intracellular pool and its appearance in the medium with a time course similar to that of TCA-precipitable material. Reverse phase HPLC demonstrated that the intracellular and medium radioactivity eluting in peak II (insulin peak) on Sephadex G-50 was composed of both intact insulin and intermediates. In conclusion, these studies demonstrated that after the internalization of insulin, rat adipocytes release not only small mol wt degradation products of insulin, but also insulin intermediates and intact insulin. The rate of retroendocytosis reported here is almost identical to the rate of insulin receptor recycling in rat adipocytes. Therefore, retroendocytosis may serve as an excellent in vitro reflection of the extent and rate of insulin receptor recycling.

Long electrocardiographic strips were analyzed from five patients who exhibited periods of typical "concealed bigeminy," i. e., recurrent unifocal extrasystoles which were separated from one another by odd numbers of normally conducted sinus beats. However, in each of these patients, there were periods in which one of three different variants of concealed bigeminy was observed. Three patients displayed an "even number" variant; i. e., there were large numbers of consecutive extrasystoles which were separated exclusively or preponderantly by even rather than by odd numbers of sinus beats. One other patient exhibited an "interpolated extrasystole" variant: those interectopic intervals which were initiated by an interpolated extrasystole contained an even number of sinus beats, whereas all other interectopic intervals contained an odd number. In the fifth patient, the distribution of the numbers of sinus beats separating extrasystoles was such as to suggest a periodic fluctuation between the classical forms of concealed bigeminy and concealed trigeminy; i. e., a "combined bigeminy and trigeminy" variant.

We are reporting 98 cases of motor evoked potential (MEP) monitoring performed between 1982 and 1986. These were divided into supratentorial, posterior fossa, and spinal cord categories. We observed in this sample that the peripheral nerve or electromyographic response was substantially more sensitive than the spinal cord response to injury and hypotension during operation and was often present bilaterally. Reversible weakening or loss of the peripheral nerve response was not associated with a deficit. However, weakening of the peripheral nerve response without recovery could warn of a motor defect after operation. The spinal cord responses can change so little in amplitude and latency with injury conditions that their reliable use during operation without accompanying peripheral nerve and muscle response monitoring may be compromised, especially in view of the often difficult recording environment in the operating room. With spinal cord monitoring, the MEP seemed closely correlated with the stresses that we imposed on the cord, as well as with subsequent clinical outcome. In posterior fossa cases, we observed sensitivity of the MEP to manipulation of the nervous system and reliable indication of the outcome in the cases monitored. Supratentorial cases present a more complex environment for monitoring; a potential pitfall is to stimulate an area that produces responses in the peripheral nerves and spinal cord, but that is not being compromised by the injury process. Alternatively, stimulating too large an area of cortex or stimulating with too high a current, which penetrates to white matter below the gray matter, may not show an injury. Although we did not encounter cases where permanent deficits were missed by the motor evoked potential, we were concerned by the appearance of temporary deficits. These may be related to technical limitations of our methods and indicate that monitoring of the supratentorial process requires substantial methodological advance for high reliability. Modulation of the MEP by prior somatosensory evoked potential stimulation seems useful and promising. Cerebellar evoked potential responses were present in humans and were sensitive to injury. Overall, this test is promising.