Preoperative evaluation of mammographic microcalcifications after neoadjuvant chemotherapy for breast cancer.

AIM: To assess the predictive value of preoperative residual mammographic microcalcifications for residual tumours after neoadjuvant chemotherapy (NAC) for breast cancer. MATERIALS AND METHODS: This single-centre retrospective study included breast cancer patients who underwent NAC and demonstrated suspicious microcalcifications within or near the tumour bed on mammography from June 2015 to August 2018. The residual microcalcifications and remnant lesion on magnetic resonance imaging (MRI) were correlated with histopathological findings of residual tumours and immunohistochemical markers. RESULTS: A total of 96 patients were included. Ten patients achieved pathological complete response (pCR) and previous suspicious microcalcifications were associated with benign pathology in 10.4% (10/96) of the patients. In the remaining 86 patients who did not achieve pCR, 61.5% (59/96) of the residual microcalcifications were associated with invasive or in situ carcinoma and 28.1% (27/96) with benign pathology. Hormone receptor-positive (HR+) patients had the highest proportion of residual malignant microcalcifications compared to HR- patients (48.9% versus 13.5%, respectively; p=0.019). MRI correlated better than residual microcalcifications on mammography in predicting residual tumour extent in all subtypes (ICC=0.709 versus 0.365). MRI also showed higher correlation with residual tumour size for the HR-/HER2+ and HR-/HER2- subtype (ICC=0.925 and 0.876, respectively). CONCLUSION: The extent of microcalcifications on mammography after NAC did not correlate with the extent of residual cancer in 38.5% of women. Regardless of the extent of microcalcifications, residual tumour extent on MRI after NAC and molecular subtype could be an accurate tool in evaluating residual cancer after NAC.

Clinical characteristics of diabetic ketoacidosis in users and non-users of SGLT2 inhibitors.

AIM: This study investigated the clinical characteristics of diabetic ketoacidosis (DKA) and compared the DKA characteristics between patients treated with and without SGLT2 inhibitors. METHODS: Data were collected from patients aged ≥ 18 years admitted for DKA at nine centres in Korea between September 2014 and April 2017. The electronic medical records of these subjects were retrospectively reviewed. Based on their history of medications taken before admission, subjects were classified as either users or non-users of SGLT2 inhibitors and their clinical characteristics of DKA were compared. RESULTS: During the study, the main subtype of DKA episodes (n = 523) was identified as type 2 diabetes (51%). Average hospitalization duration was 11 days, and average intensive care unit (ICU) time was 2.5 days. The in-hospital mortality rate was 3%, but no users of SGLT2 inhibitors died during DKA treatment. In patients taking SGLT2 inhibitors (n = 15), DKA manifested at 124 days, on average, after starting the inhibitors (range: 7-380 days). Also, SGLT2 inhibitors users had significantly lower plasma glucose levels (413 mg/dL) compared with non-users (554 mg/dL), and longer ICU stays (4 vs. 2 days; P = 0.019). CONCLUSION: In this report of recent data on the clinical features of DKA in Korea, patients using SGLT2 inhibitors needed longer treatment in ICUs compared with non-users and had lower levels of blood glucose, whereas DKA associated with SGLT2 inhibitors was rare.

Association between BMI and risk of severe hypoglycaemia in type 2 diabetes.

AIM: This study aimed to assess the association between body mass index (BMI) and the development of severe hypoglycaemia in patients with type 2 diabetes (T2D), using nationwide data for the entire South Korean population. METHODS: The association between BMI and severe hypoglycaemia was retrospectively examined from claims and National Health examination data registered between 2002 and 2015. A total of 1,366,692 subjects assigned clinical codes for T2D and prescribed antihypoglycaemic agents were included. The primary outcome was an episode of severe hypoglycaemia after the baseline health examination. RESULTS: A total of 37,682 subjects (2.7%) experienced a new severe hypoglycaemic event during the follow-up period (mean: 8.6 years). An inverse J-shaped association was observed between BMI and severe hypoglycaemic events. The association between low BMI and high risk of severe hypoglycaemia was similar in subjects who had never smoked, did not consume alcohol, did not use insulin and had no major comorbidities, after adjusting for multiple confounding variables. This association was also found to be intensified in men, young people aged 30-49 years, those with major comorbidities and insulin users. CONCLUSION: BMI and severe hypoglycaemia were found to be inversely associated. Thus, those who fall into the category of having low BMI and high risk of severe hypoglycaemia should be warned about the risk of having a hypoglycaemic event and be properly informed about hypoglycaemia to minimize the risk of fatal hypoglycaemia-related outcomes.

Lipoprotein(a) predicts a new onset of chronic kidney disease in people with Type 2 diabetes mellitus.

AIMS: We investigated the association between lipoprotein(a) [Lp(a)] level and new-onset chronic kidney disease (CKD) in patients with Type 2 diabetes. METHODS: We conducted a prospective cohort study from March 2003 to December 2004 with a median follow-up time of 10.1 years. Patients aged 25-75 years with Type 2 diabetes and without CKD [estimated glomerular filtration rate (eGFR) ≥ 90 ml/min/1.73 m(2) ) were consecutively enrolled. The eGFR was measured at least twice every year , and new-onset CKD was defined as a decreased eGFR status of < 60 ml/min/1.73 m(2) using a Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. RESULTS: Of the 862 patients who were enrolled, 560 (65.0%) completed the follow-up and 125 (22.3%) progressed to CKD. The mean age and duration of diabetes were 53.3 ± 9.6 and 7.5 ± 6.0 years, respectively. The baseline eGFR was 101.8 ± 11.3 ml/min/1.73 m(2) . After adjusting for multiple confounding factors, a Cox hazard regression analysis revealed that the third tertile of Lp(a) was significantly associated with the development of CKD during the observation period when compared with the first tertile [hazard ratio 2.12 (95% confidence interval 1.33-3.36); P = 0.001). CONCLUSIONS: In this prospective, longitudinal, observational cohort study, we demonstrated that the Lp(a) level was an independent prognostic factor for the future development of CKD in patients with Type 2 diabetes.

Antihistamines modulate the integrin signaling pathway in h9c2 rat cardiomyocytes: Possible association with cardiotoxicity.

The identification of biomarkers for toxicity prediction is crucial for drug development and safety evaluation. The selective and specific biomarkers for antihistamines-induced cardiotoxicity is not well identified yet. In order to evaluate the mechanism of the life-threatening effects caused by antihistamines, we used DNA microarrays to analyze genomic profiles in H9C2 rat cardiomyocytes that were treated with antihistamines. The gene expression profiles from drug-treated cells revealed changes in the integrin signaling pathway, suggesting that cardiac arrhythmias induced by antihistamine treatment may be mediated by changes in integrin-mediated signaling. It has been reported that integrin plays a role in QT prolongation that may induce cardiac arrhythmia. These results indicate that the integrin-mediated signaling pathway induced by antihistamines is involved in various biological mechanisms that lead to cardiac QT prolongation. Therefore, we suggest that genomic profiling of antihistamine-treated cardiomyocytes has the potential to reveal the mechanism of adverse drug reactions, and this signal pathway is applicable to prediction of in vitro cardiotoxicity induced by antihistamines as a biomarker candidate.

Endovascular management in patients with acute basilar artery obstruction: low-dose intra-arterial urokinase and mechanical clot disruption.

Mechanical clot disruption for the treatment of acute basilar artery occlusion (BAO) is known to provide a benefit. We aimed to determine the safety, recanalization rate and time-to-flow restoration of mechanical clot disruption and low dose urokinase (UK) infusions for the treatment of patients with acute BAO. Between June 2006 and June 2010, 21 patients with acute BAO underwent endovascular treatment that included angioplasty or stent placement. The time to treatment, duration of the procedure, dose of urokinase (UK), recanalization rates and symptomatic hemorrhages were analyzed. Clinical outcome measures were assessed at admission and at the time of discharge using the National Institutes of Health Stroke Scale (NIHSS) score and at three months after treatment using the modified Rankin Score (mRS). On admission, the median NIHSS score was 13.2. Median time from symptom onset to arrival at hospital was 356 minutes, and median time from symptom onset to intraarterial thrombolysis (IAT) was 49 minutes. We used the following interventional treatment regimens: Intra-arterial (IA) UK and a minimal mechanical procedure (n=14), IA UK with angioplasty (n=1), IA UK with angioplasty and stent placement (n=3) and IA UK with HyperForm (n=3). The recanalization (thrombolysis in cerebral ischemia grade II or III) rate was 90.5% (19/21). There was symptomatic hemorrhage in one patient (4.8%). The median NIHSS score at discharge was 6.3. The three-month outcome was favorable (mRS: 0-2) for 14 patients (66.7%) and poor (mRS: 3-6) for seven patients (33.3%). The overall mortality at three months was 14.3% (three patients died). Low-dose IAT with mechanical clot disruption is a safe and effective treatment for treatment for acute BAO.

Safety of ultra-rapid intravenous infusion of hepatitis B immunoglobulin in liver transplant recipients.

PURPOSE: To evaluate the safety of institutional protocol for ultra-rapid hepatitis B immunoglobulin (HBIG) infusion (10,000 IU in 30 minutes) for hepatitis B virus prophylaxis in adult liver transplant recipients. METHODS: In this case-controlled study, prospectively recruited liver transplant recipients received ultra-rapid infusions of HBIG (10,000 units in 30 minutes) for 6 months. The historical control group consisted of patients who had received 1-hour HBIG infusions (conventional rapid infusion) for the precedent 6 months. RESULTS: We found that 1472 patients had received 5744 ultra-rapid HBIG infusions, whereas 1343 patients had received 5200 conventional rapid HBIG infusions. Adverse side-effects were observed after 7 (0.13%) and 9 (0.16%) infusions, respectively (P = .763). The number of infusions per month increased significantly, from 878 ± 34 before the introduction of ultra-rapid infusion to 957 ± 29 afterwards (P < .001), an increase of 10.5%. The maximal capacity of HBIG infusions per day in the outpatient clinic increased from 53 for conventional rapid infusion to 65 for ultra-rapid infusion, without expansion of the outpatient facility or equipment. CONCLUSIONS: Nearly all adult liver recipients able to tolerate 1-hour infusions of HBIG can also tolerate ultra-rapid infusions well. Thus, it seems to be reasonable to perform ultra-rapid infusion protocol widely for patient convenience.

Bioelectrical impedance analysis for evaluation of donor hepatic steatosis in living-donor liver transplantation.

OBJECTIVE: To assess whether bioelectrical impedance analysis (BIA) can be used to evaluate the degree of hepatic steatosis in potential living liver donors. MATERIAL AND METHODS: From May 2008 to April 2009, BIA was measured in 302 living donor candidates. Correlations among body mass index (BMI; calculated as weight in kilograms divided by height in meters squared), total fatty changes at percutaneous needle liver biopsy, and BIA-derived fat composition were assessed. RESULTS: The median (range) BIA-derived fat proportion was 19.4% (4.8%-35.3%), BMI was 24 (17-39), and hepatic steatosis at liver biopsy was 2% (0%-75%). Crude correlations were observed between BIA-derived fat proportion and hepatic steatosis (r(2) = 0.14; P = .000), between BMI and hepatic steatosis (r(2) = 0.27; P = .000), and between BMI and BIA-derived fat proportion (r(2) = .25; P = .000). Receiver operating characteristic curve analysis revealed that the area under the curve of BIA-derived fat proportion was smaller than that of BMI, and no significant cutoff value was identified. CONCLUSIONS: These results suggest that BIA-derived fat composition alone cannot be used to accurately determine the degree of hepatic steatosis. However, a combination of BMI and BIA-derived fat composition may increase clinical ability to assess hepatic steatosis.

Batch and repeated batch production of L (+)-lactic acid by Enterococcus faecalis RKY1 using wood hydrolyzate and corn steep liquor.

Lactic acid production was investigated for batch and repeated batch cultures of Enterococcus faecalis RKY1, using wood hydrolyzate and corn steep liquor. When wood hydrolyzate (equivalent to 50 g l(-1) glucose) supplemented with 15-60 g l(-1) corn steep liquor was used as a raw material for fermentation, up to 48.6 g l(-1) of lactic acid was produced with, volumetric productivities ranging between 0.8 and 1.4 g l(-1 )h(-1). When a medium containing wood hydrolyzate and 15 g l(-1) corn steep liquor was supplemented with 1.5 g l(-1) yeast extract, we observed 1.9-fold and 1.6-fold increases in lactic acid productivity and cell growth, respectively. In this case, the nitrogen source cost for producing 1 kg lactic acid can be reduced to 23% of that for fermentation from wood hydrolyzate using 15 g l(-1) yeast extract as a single nitrogen source. In addition, lactic acid productivity could be maximized by conducting a cell-recycle repeated batch culture of E. faecalis RKY1. The maximum productivity for this process was determined to be 4.0 g l(-1 )h(-1).

Expression and relationship of the insulin-like growth factor system with posthatch growth in the Korean Native Ogol chicken.

Insulin-like growth factors (IGF) act as regulators that modulate proliferation and differentiation of various cells. Also, IGF are involved in metabolism and body growth by regulating the synthesis and degradation of glycogen and proteins in animals. However, the effect of IGF system on body growth in poultry including Korean Native Ogol chickens (KNOC) has not been thoroughly studied. Therefore, this study was performed to investigate the expressions of IGF system and the relationship of IGF with body growth during posthatch growth in KNOC. Sera and organs were collected at hatch and at 1, 3, 5, and 7 wk. The mRNA expressions of IGF, IGF-I receptor, and IGF binding protein (IGFBP)-2 were quantitatively analyzed by reverse transcription (RT)-PCR. The IGF concentrations were measured by heterologous RIA, and the expression of IGFBP-2 was detected by Western ligand blotting. The body weight of KNOC rapidly increased during the experimental period, and increase in breast muscle weight was 5-fold from 1 to 3 wk. Although the circulating IGF-I concentration gradually increased, the level of IGF-I in breast muscle rapidly declined during growth period. The IGF-II expression was not similar to IGFBP-2 during postnatal growth. Moreover, the breast muscle IGF-II concentration was mainly correlated with body growth at 7 wk and breast muscle IGF-I at 1 and 5 wk. Taken together, the present study suggested that the endocrine manner of IGF-I was more important than auto/paracrine actions in body growth of KNOC and that expression of IGF-II was involved in body growth and IGF-I during posthatch growth of KNOC.

Inhibitory effects of MK-801 on contextual sensitization to climbing behavior and on development of tolerance to hypothermia induced by a single high dose of apomorphine.

A single high dose of apomorphine (10 mg x kg(-1)) produced not only contextual sensitization to and conditioning of climbing behavior, but also context-independent tolerance to hypothermia. MK-801 (0.15 and 0.3 mg x kg(-1)) inhibited contextual sensitization to and conditioning of climbing behavior. Development of tolerance to hypothermia was also inhibited by MK-801. Dopamine D1 antagonist, SCH23390 (0.5 mg x kg(-1)), but not D2 antagonist, sulpiride, inhibited sensitization to and conditioning of climbing behavior. D2 antagonist, sulpiride (50 mg x kg(-1)), but not D1 antagonist, SCH23390, inhibited development of tolerance to hypothermia. These results suggest that MK-801 inhibited contextual sensitization to climbing behavior and development of tolerance to hypothermia through glutamatergic modulation of dopaminergic functions at dopamine receptors.

Mechanisms and kinetics of high-temperature cadmium sorption by packed bed of calcined kaolin.

Sorption experiments by passing CdCl2-carrying flue gas through the packed bed of calcined macro-porous kaolin particles were performed over a temperature range of 973-1173K and a CdCl2 partial pressure range of 8-16.1 Pa. The observed structural change of the sorbent mineral at the stage of sorption and the results of desorption tests revealed the characteristics of an irreversible chemical reaction as a major cadmium capturing mechanism. In the fully saturated kaolin sorbent, CdO x Al2O x 2SiO2 is present as a sorption reaction product together with a smaller amount of 2CdO x Al2O x 2SiO2. The increase in sorbent bed temperature resulted in an increase in the rate of sorption, but it had no effect on maximum cadmium uptake. The gas-phase CdCl2 diffusion into the macro pores of calcined kaolin had a negligible effect on the overall sorption rate. The reaction between gaseous CdCl2 and solid Al2O3 x 2SiO2 is very sensitive to the concentration of CdCl2 but relatively insensitive to the temperature of the sorbent bed. The order of reaction with respect to the CdCl2 vapor concentration was determined to be 3.26. The activation energy, Ea, was estimated as 5.56 kcal/mol according to the Arrhenius relationship.

Inhibition of baclofen on morphine-induced hyperactivity, reverse tolerance and postsynaptic dopamine receptor supersensitivity.

The effects of baclofen on the development of reverse tolerance and postsynaptic dopamine receptor supersensitivity induced by morphine were examined in mice. A single administration of morphine induced hyperactivity and the morphine-induced hyperactivity was inhibited dose dependently by the administration of a GABA(B)receptor agonist, baclofen (1.25, 2.5 and 5 mg kg(-1), i.p.). Daily repeated administration of morphine developed reverse tolerance to the hyperactivity of morphine. The concomitant administration of baclofen inhibited the morphine-induced hyperactivity and the baclofen administration prior to and during the chronic administration of morphine in mice inhibited the development of reverse tolerance to the hyperactivity of morphine (10 mg kg(-1), s.c.). Postsynaptic dopamine receptor supersensitivity was also developed in reverse-tolerant mice that had received the same morphine. The development of postsynaptic dopamine receptor supersensitivity was evidenced by the enhanced ambulatory activity of apomorphine (2 mg kg(-1), s.c.). Baclofen also inhibited the development of postsynaptic dopamine receptor supersensitivity induced by the chronic administration of morphine. These results suggest that the hyperactivity, reverse tolerance and postsynaptic dopamine receptor supersensitivity induced by morphine may be modulated via the activation of GABA(B)receptors induced by baclofen.

The use of transgenic mice to analyze the role of accessory factor two in the regulation of phosphoenolpyruvate carboxykinase (GTP) gene transcription during diabetes.

The regulation of transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C) (4.1.1.32) during diabetes is a complex process that involves a number of regulatory elements in the PEPCK-C gene promoter. The accessory factor 2 (AF2)-binding region that is contained within the glucocorticoid regulatory unit of the PEPCK-C gene promoter (-451 to -353) has been implicated in the action of both insulin and glucocorticoids on PEPCK-C gene transcription. To determine the role of AF2 in these processes, we have generated a mouse model bearing a transgene that contains the PEPCK-C gene promoter with a mutation in the AF2-binding region. This promoter is linked to the structural gene for human growth hormone that is biologically inactive (AF2-2000/hGx). In the absence of the AF2 regulatory element, the transcription of the transgene in the liver is not induced by diabetes but is inhibited by the administration of insulin. There is also a marked reduction in the response of the AF2-2000/hGx gene in the kidney to the administration of glucocorticoids. The AF2-2000/hGx gene in the liver responds normally to a high carbohydrate diet with a marked decrease in gene transcription. This suggests that insulin is not exerting its usual negative effect on the PEPCK-C gene promoter through the AF2 site. In contrast, the response of this transgene to a high fat/carbohydrate-free diet is severely blunted. Our results support a role for the AF2 site in the PEPCK-C gene promoter in the effect of glucocorticoids, but not insulin, on PEPCK-C gene transcription in the liver.

Clinical predictors of mental disorders among medical outpatients.

BACKGROUND: Mental disorders are common among primary care patients and often not detected by primary care physicians. We report on clinical cues that may allow physicians to target patients for psychiatric screening. METHODS: Two hundred fifty consecutive adults presenting to a walk-in clinic completed previsit surveys assessing demographics, symptom characteristics, recent stress, functional status (Medical Outcomes Study Short Form-6), and mental disorders (Primary Care Evaluation of Mental Disorders [PRIME-MD]). Patients with positive findings for a mental disorder on the PRIME-MD underwent a semistructured interview. Immediately after the visit, physicians completed the Difficult Doctor Patient Relationship Questionnaire. RESULTS: Patients averaged 50.5 years of age (range, 18-92 years). Little more than half were women (53%); 43%, white; 44%, African American; 8%, Hispanic; and 6%, other. Twenty-six percent had an underlying mental disorder; 11% had more than 1 mental disorder. Sixteen percent had a depressive disorder; 6%, major depression; 11%, an anxiety disorder; 2%, panic disorder; and 9%, a somatoform disorder. Independent correlates of a mental disorder included reporting recent stress (odds ratio [OR], 6.7; 95% confidence interval [CI], 3.3-13.6), having 5 or more physical symptoms (OR, 4.0; 95% CI, 2.1-7.9), or reporting health to be less than very good (OR, 2.2; 95% CI, 1.1-4.3). There was a stepwise increase in the likelihood of having a mental disorder and number of correlates present. Among patients with no predictors, only 2% had an underlying mental disorder, compared with 72% among patients with all 3 clinical predictors. CONCLUSIONS: Patients who report recent stress, 5 or more physical symptoms, or poor health are more likely to have an underlying mental disorder. These clinical cues may allow clinicians to select patients in whom formal screening for mental disorders would be particularly fruitful.

Simultaneous determination of delta-aminolevulinic acid, porphobilinogen, levulinic acid and glycine in culture broth by capillary electrophoresis.

Capillary electrophoretic simultaneous determination of a mixture containing delta-aminolevulinic acid, porphobilinogen, levulinic acid and glycine was investigated. With increases in the sodium tetraborate buffer concentration (5-70 mM), resolution of the four components was improved, but the migration time was increased. Alternatively, with increases in the applied voltage (5-22.5 kV), a shortened migration time was seen but this adversely affected resolution. The components were separated with high resolution by using a fused-silica capillary column (75 cm x 75 microm I.D.) filled with 30 mM sodium tetraborate buffer (pH 9.3-9.4) under the applied voltage of 20 kV (constant voltage mode). When the established method was applied to the culture broth of Rhodopseudomonas sphaeroides, a photosynthetic bacterium, the four components mentioned above were separated with good resolution. Furthermore, the use of this method would provide a fast, sensitive and specific method for monitoring the administration of delta-aminolevulinic acid in photodynamic cancer therapy, for the measurement of delta-aminolevulinic acid dehydratase activity in erythrocytes, and for testing the delta-aminolevulinic acid assay and for impurities in drug formulation.

Determinants of the cardiomyopathic phenotype in chimeric mice overexpressing cardiac Gsalpha.

Mice with overexpressed cardiac Gsalpha develop cardiomyopathy, characterized by myocyte hypertrophy and extensive myocardial fibrosis. The cardiomyopathy likely involves chronically enhanced beta-adrenergic signaling, because it can be blocked with long-term propranolol treatment. It remains unknown whether the genotype of the myocyte is solely responsible for the progressive pathological changes. A chimeric population in the heart should answer this question. Accordingly, we developed a chimeric animal, which combined cells from a transgenic overexpressed Gsalpha parent and a Rosa mouse containing the LacZ reporter gene, facilitating identification of the non-Gsalpha cells, which express a blue color with exposure to beta-galactosidase. We studied these animals at 14 to 17 months of age (when cardiomyopathy should have been present), with the proportion of Gsalpha cells in the myocardium ranging from 5% to 88%. beta-Galactosidase staining of the hearts demonstrated Gsalpha and Rosa cells, exhibiting a mosaic pattern. The fibrosis and hypertrophy, characteristic of the cardiomyopathy, were not distributed randomly. There was a direct correlation (r=0.85) between the extent of myocyte hypertrophy (determined by computer imaging) and the quantity of Gsalpha cells. The fibrosis, determined by picric acid Sirius red, was also more prominent in areas with the greatest Gsalpha cell density, with a correlation of r=0.88. Thus, the overexpressed Gsalpha can exert its action over the life of the animal, resulting in a local picture of cardiomyopathic damage in discrete regions of the heart, where clusters of the overexpressed Gsalpha cells reside, sparing the clusters of normal cells derived from the normal Rosa parent.

Bioconversion of fumarate to succinate using glycerol as a carbon source.

In this study, a facultative bacterium that converts fumarate to succinate at a high yield was isolated. The yield of bioconversion was enhanced about 1.2 times by addition of glucose into culture medium at an initial concentration of 6 g/L. When the initial cell density was high (2 g/L), the succinate produced at pH 7.0 for initial fumarate concentrations of 30, 50, 80, and 100 g/L were 29.3, 40.9, 63.6, and 82.5 g/L, respectively, showing an increase with the initial fumarate concentration. The high yield of 96.8%/mole of fumarate in just 4 h was obtained at the initial fumarate concentration of 30 g/L. Comparing these values to those obtained with low cell culture (0.2 g/L), we found that the amount of succinate produced was similar, but the production rate in the high cell culture was about three times higher than was the case in the low cell culture. This strain converted fumarate to succinate at a rate of 3.5 g/L.h under the sparge of CO2.

Differential in vivo activities of bovine growth hormone analogues.

In rodents, bovine (b) growth hormone (GH) binds only to GH receptors, while human (h) GH binds to both GH and PRL receptors. The phenotypic consequences of expression of bGH and hGH in transgenic mice are different and, in some cases, opposite. In the present study, site-directed in vitro mutagenesis of the bGH gene was used systematically to eliminate its differences from hGH at one, two, three or four suspected of conferring lactogenic activity: D11, H18, S57 and T60, respectively (corresponding to sites 12, 19, 57 and 60 of the bGH molecule). The resulting bGH analogues were expressed in cell lines and in transgenic mice. All of the seven bGH analogues produced retained their ability to bind to GH receptors and exhibited somatogenic activity in vitro and in vivo. However, none of them were able to bind to PRL receptors or to elicit detectable lactogenic response in vitro. Transgenic animals expressing any of the generated analogues were characterized by gigantism and splanchnomegaly. The effects of expression of each of the double, triple or quadruple mutants on the seminal vesicle weight resembled the effects of wild-type hGH and differed from the effects of expression of wild-type bGH. There were differences between the effects of the expression of different bGH analogues on plasma PRL levels and on the PRL response to pharmacological blockade of catecholamine synthesis. Plasma LH levels in ovariectomized females were suppressed by several of the analogues tested, an effect not seen in animals expressing wild-type bGH or hGH. Dopamine turnover in the median eminence of male mice was also altered in animals expressing different bGH analogues but not in those expressing wild-type bGH or hGH. In ovariectomized females, the effects of different bGH analogs on the turnover of dopamine and norepinephrine in the median eminence included changes resembling those detected in animals expressing hGH, as well as alterations differing from the effects of both bGH and hGH. The results indicate that biological actions of these bGH analogues cannot be characterized simply in terms of enhanced or reduced somatogenic or lactogenic activity and raise a possibility that different sites, domains or features of tri-dimensional structure of GH are involved in its actions on different cellular targets.

Phosphoenolpyruvate carboxykinase (GTP) gene transcription and hyperglycemia are regulated by glucocorticoids in genetically obese db/db transgenic mice.

The molecular mechanisms underlying increased hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene transcription and gluconeogenesis in type II diabetes are largely unknown. To examine the involvement of glucocorticoids and the cis-acting insulin response sequence (IRS, -416/-407) in the genetically obese db/db mouse model, we generated crosses between C57BL/KsJ-db/+ mice and transgenic mice that express -460 or -2000 base pairs of the rat PEPCK gene promoter containing an intact or mutated IRS, linked to a reporter gene. Transgenic mice expressing the intact PEPCK(460)-CRP (C-reactive protein) transgene bred to near homozygosity at the db locus were obese, hyperinsulinemic, and developed fasting hyperglycemia (389 +/- 26 mg/100 ml) between 4 and 10 weeks of age. Levels of CRP reporter gene expression were increased 2-fold despite severe hyperinsulinemia compared with non-diabetic non-obese transgenic mice. Reporter gene expression was also increased 2-fold in transgenic obese diabetic db/db mice bearing a mutation in the IRS, -2000(IRS)-hGx, compared with non-obese non-diabetic transgenic 2000(IRS)-hGx mice. Treatment of obese diabetic db/db transgenic mice with the glucocorticoid receptor blocker RU 486 decreased plasma glucose by 50% and reduced PEPCK, GLUT2, glucose-6-phosphatase, tyrosine aminotransferase, CRP, and hGx reporter gene expression to levels similar to those of non-obese normoglycemic transgenic mice. Taken together, these results establish that -460 bp of 5'-flanking sequence is sufficient to mediate the induction of PEPCK gene transcription in genetically obese db/db mice during the development of hyperglycemia. The results further demonstrate that the mechanism underlying increased expression of gluconeogenic enzymes in the db/db mouse requires the action of glucocorticoids and occurs independently of factors acting through the PEPCK IRS (-416/-407) promoter binding site.