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Introduction: Ductoscopy is a low invasive method enabling the diagnostics of intraductal proliferative lesions in breasts. Fiberoptic ductoscopy (FDS) is important in the diagnosis of patients with pathological nipple discharge. There are attempts to apply FDS in patients with breast cancer without the presence of nipple discharge., Aim: To assess fiberoptic ductoscopy in the diagnostics of breast neoplasms., Material and Methods: The material was composed of a group of 164 patients treated for intraductal proliferative lesions in breasts. In the analyzed group of patients, FDS was conducted in 128 patients with pathological nipple discharge and 36 patients with the presence of breast cancer. The analyzed period was divided into three sub-periods. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of FDS examination verified by post-operative histopathological examination were analyzed. The safety of the method was also assessed, taking into consideration the complications., Results: An increasing number of successful ductoscopies together with the number of performed FDS examinations was noted. There were statistically significant differences in the percentage of successful cannulations in relation to the number of performed FDS examinations in the three subsequent stages of the project (p = 0.011). The duration of FDS examination in the third period was reduced in comparison with the first and second period (p < 0.001). Sensitivity of fiberoptic ductoscopy is 68.1%, specificity 77.3% and PPV 90.4%, but NPV is 44.1%., Conclusions: The introduction of fiberoptic ductoscopy in our clinic has contributed to the widening of the diagnostic possibilities of small intraductal lesions of the mammary gland.

Olaparib (AZD2281) is an oral poly(ADP-ribose) polymerase (PARP) inhibitor with antitumour activity in cancer patients with BRCA1/2 germline mutations and in patients with homologous recombination deficiency. In this dose-finding study, patients were randomized to olaparib 10, 30, 100, 200 or 400 mg (capsule formulation) twice daily for the 4-5 days preceding breast cancer surgery. The primary objective was to identify an effective biological dose of olaparib for future trials. Secondary endpoints included evaluation of PARP-1 inhibition dose/exposure-response, and safety. Olaparib plasma pharmacokinetics (PK) and the pharmacodynamics (PD) in tumour and peripheral blood mononuclear cells (PBMCs) were evaluated. Population PK/PD modelling was performed on pooled data from this study and a previously reported study. Sixty patients were randomized (n = 12, each dose). Dose-dependent increases in exposure to olaparib were observed, but at ~50 % lower plasma exposure levels than seen in advanced disease studies. The mean maximal extent of PARP inhibition in PBMCs and tumour tissue was 50.6 % and 70.0 %, respectively, and was similar to inhibitory levels reported previously. No PARP inhibition-dose relationship was observed. Due to the unexpectedly low olaparib exposure, we were unable to determine an effective biological dose. Common adverse events included procedural pain (n = 31 patients), nausea, asthenia, malaise and increased blood creatinine (n = 6, each); these were of mild-to-moderate intensity, and all were manageable. Despite low olaparib exposure, PARP inhibition was consistent with previous reports. Reasons for the inter-study differences in exposure are unclear. The tolerability profile of olaparib was consistent with previous studies.

Introduction: The aim of the work was to analyze the impact of selected factors on the incidence of seroma formation in breast cancer patients undergoing mastectomy., Material and Methods: One hundred and fifty breast cancer patients were prospectively enrolled in the study. All patients had mastectomy performed using the same operative technique with electrocoagulation. The amount of seroma formed after surgery and its duration were correlated with selected demographic, clinical and pathological parameters., Results: The cumulative total seroma volume collected by the end of treatment was higher and the overall time of seroma treatment was longer in patients over the age of 60 years (p = 0.001 and p = 0.001 respectively). Duration of seroma was significantly longer in obese patients (p = 0.036). The cumulative total seroma volume collected by the end of treatment was higher and the overall time of seroma treatment was longer in patients who had over 130 ml of lymph drained during the first 24 postoperative hours (p < 0.001 and p = 0.001 respectively). Additionally, longer duration of seroma was observed in patients with pathological stage I and II according to TNM-UICC (p = 0.042) and in patients with ≥ 1200 g weight resected of mammary gland (p = 0.05)., Conclusions: Age and obesity are important prognostic factors influencing seroma formation in breast cancer patients undergoing mastectomy. The amount of lymph formed during first postoperative day may have predictive value in assessing cumulative total seroma volume collected during treatment and its overall duration.

Background: Previous studies showed the prognostic and predictive impact of human epidermal growth factor receptor 2 (HER-2) gene alterations analyzed separately and jointly with topoisomerase II α (TOP2A) gene alterations; however, the role of TOP2A gene abnormalities alone has not been thoroughly investigated. Additionally, TOP2A aberrations were typically studied in HER-2-positive (HER-2(+)) tumors because these genes are frequently coamplified. Therefore, the knowledge concerning the impact of TOP2A abnormalities in HER-2-negative (HER-2(-)) patients is scarce. This study aimed to investigate the clinical significance of TOP2A anomalies in breast cancer patients with HER-2(-) and HER-2(+) tumors., Materials and Methods: Snap-frozen tumor samples from 322 consecutive stage I-III breast cancer patients were analyzed for TOP2A gene dosage using quantitative real-time PCR (qPCR)., Results: A high TOP2A gene dosage was found in 94 tumors (29%)-32% and 27% of HER-2(+) and HER-2(-) tumors, respectively. The mean TOP2A gene dosages in the HER-2(+) and HER-2(-) groups were 1.49 ± 1.03 and 1.09 ± 0.35, respectively. High TOP2A gene dosage had an inverse prognostic impact in terms of shorter disease-free survival (DFS) and overall survival (OS) times in the entire group and in both the HER-2(-) and HER-2(+) subgroups. The unfavorable prognostic impact of TOP2A gene dosage was maintained in the multivariate Cox regression analysis in the entire group and in HER-2(-) patients., Conclusions: A high gene dosage of TOP2A determined using qPCR occurs frequently both in HER-2(+) and HER-2(-) tumors and has a strong adverse prognostic impact.

Background: In this prospective, randomized, placebo-controlled, double-blinded clinical trial we tested the hypothesis that preemptive analgesia with bupivacaine applied in the area of the surgical incision in patients undergoing mastectomy for breast cancer would reduce post-operative acute pain and would reduce the amount of analgesics used during surgery and in the post-operative period., Material/methods: Participants were assigned into 1 of 2 groups--with bupivacaine applied in the area of surgical incision or with placebo. We assessed the intraoperative consumption of fentanyl, the postoperative consumption of morphine delivered using a PCA method, and the subjective pain intensity according to VAS score reported by patients in the early post-operative period., Results: Out of 121 consecutive cases qualified for mastectomy, 112 women were allocated randomly to 1 of 2 groups--group A (bupivacaine) and group B (placebo). The final study group comprised 106 breast cancer cases. Between the groups, a statistically significant difference was observed with respect to: lower fentanyl consumption during surgery (p = 0.011), lower morphine (delivered by means of a PCA) consumption between the 4-12th postoperative hours (p = 0.02) and significantly lower pain intensity assessed according to VAS score at the 4th and 12th hours after surgery (p = 0.004 and p = 0.02 respectively) for the group A patients., Conclusions: Preemptive analgesia application in the form of infiltration of the area of planned surgical incisions with bupivacaine in breast cancer patients undergoing mastectomy decreases post-operative pain sensation, limits the amount of fentanyl used during surgery, and reduces the demand for opiates in the hours soon after surgery.

Background: Although studies comparing the surgical treatment of gastric carcinoma in Japan and Western industrialized countries have revealed differing survival rates, no studies to date have been performed comparing Western and Eastern Europe. This study aimed to compare demographics and surgical practice as well as the related prognostic impact on gastric cancer patients treated in Poland and Germany., Methods: This retrospective study included gastric cancer patients treated between 1999 and 2004 by surgical departments in Gdansk (Poland) and Cologne (Germany). Univariate and multivariate analyses of demographic, histopathological, surgical, and prognostic data were performed., Results: Included were 117 patients from Gdansk and 130 patients from Cologne. The Cologne patients showed higher incidence rates of serious comorbidity, pT1 cancer, and distant metastasis than those from Gdansk. Indications for and frequency of selected surgical procedures differed significantly. D2-lymphadenectomy was performed in 89% of the Cologne patients, while D1-lymphadenectomy was done for 85% of the Gdansk patients. Univariate analysis yielded a 5-year survival rate of 28.3% for the Gdansk patients, and 40.3% for the Cologne patients (p = 0.056). Independent prognostic factors were pT category (p = 0.002), pN category (p < 0.001), pM category (p = 0.027), residual tumor (R) category (p = 0.004), age (p = 0.012), and number of resected lymph nodes (p = 0.005)., Conclusions: Significant differences of clinical and surgical parameters exist between gastric cancer patients treated in Poland and Germany. In addition to established independent prognostic factors, we found that survival improved with each additionally resected lymph node.

We describe simultaneous surgery performed on a 71-year-old woman with critical aortic stenosis and gastric cancer that were diagnosed at the same time. The patient qualified for simultaneous surgery for both these diseases. Good early outcome was achieved. There is a lack of standards for treatment of patients with coexistence of two life-threatening conditions. We discuss surgical tactics and potential benefits of such management.

Aims: The aim of the study was to assess the long-term outcomes of the surgical-only management of gastric cancer (GC) patients treated in a single centre over 10-year span preceding introduction of multimodal therapy to the clinical practice., Methods: Two hundred and one patients with pathologic confirmation of GC treated for resectable tumor mostly with curative intent were enrolled in the study. The analysis comprised a review of the medical histories, descriptions of the operations, results of histopathological studies and long-time survival., Results: Median survival time and 5-year survival rate for patients with R0 vs. R1 and R2 resection were 3.85 years and 43.5% versus 0.86 years and 7.8%, respectively. Univariate analyses showed that 5-year survival rates were correlated with histopathological type according to Laurén, type of lymphadenectomy performed, spleen preservation, stage of disease and the degree of radical resection. Independent prognostic factors in multivariate analysis were: no residual tumor after resection, type of lymphadenectomy, depth of tumor invasion in the gastric wall and lymph nodes status., Conclusions: Retrospective analysis of the long-term results of the surgical-only management of GC patients may be useful in assessment of the quality of medical services, especially prior to introduction of multimodal therapy., (Copyright © 2010 S. Karger AG, Basel.)

Branched-chain alpha-ketoacid dehydrogense complex (BCKDH) is a regulatory enzyme of valine, isoleucine and leucine catabolism. Its activity is mainly regulated by covalent modification achieved by a specific BCKDH kinase (BDK) and phosphatase (BDP). The goal of our study was to investigate the effect of increasing doses of fenofibrate on BDK and BCKDH activities in rat's liver. For 14 days fenofibrate was administrated to Wistar male rats (fed chow containing 8% protein) at one of the daily doses: 5, 10, 20 and 50mg/kg. Control group was given only vehicle (0.3% methylcellulose). BDK activity as well as actual BCKDH activity and total BCKDH activity were assayed spectrophotometrically and BDK protein amount was determined by Western blotting. In rats administered fenofibrate BDK activity decreased by 61%, 64%, 66% and 89% (p<0.0001). Changes in BDK protein expression did not correspond with changes in BDK activity. BCKDH complex actual activity was 3.7+/-0.3, 4.1+/-0.1, 4.6+/-0.3 and 4.0+/-0.3fold higher (p<0.0001) and BCKDH total activity 1.3+/-0.1, 1.3+/-0.1, 1.5+/-0.1 and 1.3+/-0.1fold higher comparing to control group (p<0.001). BCKDH activity state (percentage of active, dephosphorylated form) increased 2.8+/-0.2, 3.1+/-0.1, 3.2+/-0.1 and 3.0+/-0.1fold (p<0.0001). In addition, fenofibrate prevented body weight gain starting from the dose of 10mg/kg/day and induced hepatomegaly in a dose-dependent manner. It can be concluded that fenofibrate under condition of protein restriction starting from the lowest dose inhibits BDK activity at the posttranslational level and increases BCKDH activity state. It is conceivable that fenofibrate decreases of branched-chain amino acids (BCAA) levels by stimulation of their catabolism. Since leucine plays an important role in up-regulation of protein anabolism in muscles, the reduced level of this amino acid may be one of the factors involved in pathomechanism of myopathy observed during treatment with fenofibrate.

Purpose: The aim of this work is to compare selected parameters of implants and natural dose volume histograms for two techniques of interstitial pulsed dose rate brachytherapy (PDR BT) as a boost to the tumour bed in breast-conserving therapy (BCT)., Material and Methods: Data of T 1-3 N 0-2 M 0 breast cancer patients who underwent BCT with BT boost between 05.2002 and 12.2008 were analysed. Ninety two patients were implanted with rigid tubes after breast irradiation (group A) and 96 had a peri-operative BT with an intra-operative flexible tube placement and subsequent whole breast radiotherapy (group B). In both groups PDR BT of 15 Gy (1 Gy/pulse/h) was administered based on Paris system rules, and volume optimization using BT planning system PLATO., Results: Three-plane implant was used in 62% and 8% of patients in group A and B, respectively, and two-plane implant in 38% of group A and in 84% of group B, with a median of 11 and 9 tubes respectively. The average volume for the prescribed dose (V 100 ) was 42.0 ± 25.4 cc (group A) and 34.1 ± 19.7 cc (group B), respectively (p = 0.017). The individual V 50 and V 200 were similar. Quality index (QI) was not impacted by the technique of BT (mean QI was 1.80 ± 0.10 and 1.75 ± 0.46 for the groups A and B, respectively). Uniformity index (UI) in respective groups was 1.60 ± 0.10 and 1.52 ± 0.21 (p = 0.001)., Conclusions: Implant volume encompassed by prescribed dose was significantly lower with intra-operative plastic tubes placement. In respect to the QI, these two BT techniques were comparable. The target volume coverage by the dose distribution as defined by UI was better for rigid tubes.

Background: Hormone replacement therapy (HT) is known to increase the risk of breast cancer in healthy women, but its effect on breast cancer risk in breast cancer survivors is less clear. The randomized HABITS study, which compared HT for menopausal symptoms with best management without hormones among women with previously treated breast cancer, was stopped early due to suspicions of an increased risk of new breast cancer events following HT. We present results after extended follow-up., Methods: HABITS was a randomized, non-placebo-controlled noninferiority trial that aimed to be at a power of 80% to detect a 36% increase in the hazard ratio (HR) for a new breast cancer event following HT. Cox models were used to estimate relative risks of a breast cancer event, the maximum likelihood method was used to calculate 95% confidence intervals (CIs), and chi(2) tests were used to assess statistical significance, with all P values based on two-sided tests. The absolute risk of a new breast cancer event was estimated with the cumulative incidence function. Most patients who received HT were prescribed continuous combined or sequential estradiol hemihydrate and norethisterone., Results: Of the 447 women randomly assigned, 442 could be followed for a median of 4 years. Thirty-nine of the 221 women in the HT arm and 17 of the 221 women in the control arm experienced a new breast cancer event (HR = 2.4, 95% CI = 1.3 to 4.2). Cumulative incidences at 5 years were 22.2% in the HT arm and 8.0% in the control arm. By the end of follow-up, six women in the HT arm had died of breast cancer and six were alive with distant metastases. In the control arm, five women had died of breast cancer and four had metastatic breast cancer (P = .51, log-rank test)., Conclusion: After extended follow-up, there was a clinically and statistically significant increased risk of a new breast cancer event in survivors who took HT.

The PDC (pyruvate dehydrogenase complex) is strongly inhibited by phosphorylation during starvation to conserve substrates for gluconeogenesis. The role of PDHK4 (pyruvate dehydrogenase kinase isoenzyme 4) in regulation of PDC by this mechanism was investigated with PDHK4-/- mice (homozygous PDHK4 knockout mice). Starvation lowers blood glucose more in mice lacking PDHK4 than in wild-type mice. The activity state of PDC (percentage dephosphorylated and active) is greater in kidney, gastrocnemius muscle, diaphragm and heart but not in the liver of starved PDHK4-/- mice. Intermediates of the gluconeogenic pathway are lower in concentration in the liver of starved PDHK4-/- mice, consistent with a lower rate of gluconeogenesis due to a substrate supply limitation. The concentration of gluconeogenic substrates is lower in the blood of starved PDHK4-/- mice, consistent with reduced formation in peripheral tissues. Isolated diaphragms from starved PDHK4-/- mice accumulate less lactate and pyruvate because of a faster rate of pyruvate oxidation and a reduced rate of glycolysis. BCAAs (branched chain amino acids) are higher in the blood in starved PDHK4-/- mice, consistent with lower blood alanine levels and the importance of BCAAs as a source of amino groups for alanine formation. Non-esterified fatty acids are also elevated more in the blood of starved PDHK4-/- mice, consistent with lower rates of fatty acid oxidation due to increased rates of glucose and pyruvate oxidation due to greater PDC activity. Up-regulation of PDHK4 in tissues other than the liver is clearly important during starvation for regulation of PDC activity and glucose homoeostasis.

Clofibrate promotes catabolism of branched-chain amino acids by increasing the activity of the branched-chain alpha-keto acid dehydrogenase [BCKDH] complex. Depending upon the sex of the rats, nutritional state, and tissue being studied, clofibrate can affect BCKDH complex activity by three different mechanisms. First, by directly inhibiting BCKDH kinase activity, clofibrate can increase the proportion of the BCKDH complex in the active, dephosphorylated state. This occurs in situations in which the BCKDH complex is largely inactive due to phosphorylation, e.g., in the skeletal muscle of chow-fed rats or in the liver of female rats late in the light cycle. Second, by increasing the levels at which the enzyme components of the BCKDH complex are expressed, clofibrate can increase the total enzymatic activity of the BCKDH complex. This is readily demonstrated in livers of rats fed a low-protein diet, a nutritional condition that induces a decrease in the level of expression of the BCKDH complex. Third, by decreasing the amount of BCKDH kinase expressed and therefore its activity, clofibrate induces an increase in the percentage of the BCKDH complex in the active, dephosphorylated state. This occurs in the livers of rats fed a low-protein diet, a nutritional condition that causes inactivation of the BCKDH complex due to upregulation of the amount of BCKDH kinase. WY-14,643, which, like clofibric acid, is a ligand for the peroxisome-proliferator-activated receptor alpha [PPARalpha], does not directly inhibit BCKDH kinase but produces the same long-term effects as clofibrate on expression of the BCKDH complex and its kinase. Thus, clofibrate is unique in its capacity to stimulate BCAA oxidation through inhibition of BCKDH kinase activity, whereas PPARalpha activators in general promote BCAA oxidation by increasing expression of components of the BCKDH complex and decreasing expression of the BCKDH kinase.

Night-float rotations were designed to alleviate the workload of residents on night call and thereby improve patient safety. However, the impact of the night float on residents is yet to be surveyed. We assessed the impact of the night-float rotation on pediatric residents using an anonymous questionnaire that covered topics, based on recall, about sleep, mood, alertness, adjustment, and others. The study was conducted in a major tertiary pediatric teaching hospital in the United States. Participants were pediatric residents who had completed one or two night-float rotations and were in active training at our teaching hospital at the time of the study. Fifty-two of 60 eligible residents (87%) responded. Sleep duration during the night-float rotation was shorter than during day-shift work in 24 residents (46%), longer in 20 (38%), and unchanged in eight (15%). A higher proportion of residents took longer to fall asleep, had more difficulty falling asleep, had more sleep interruptions, and felt less rested upon awakening. Twenty-four residents (46%) felt that their bodies never adjusted to the night shift. Also, 22 residents (43%) felt moody or depressed in contrast to seven (14%) who felt depressed during the daytime rotation (p = 0.0001). Twenty-one residents (41%) felt they were slower in their thinking during the night float than daytime rotations. The results suggest that disturbances of sleep and mood and decreased alertness, typical of night shift, are present in the night-float rotation. Residency programs should monitor closely the impact of the night-float rotation on resident well being and patient safety. The impact of night-shift work should be considered in the design of night-float schedules, and teaching should be provided for residents to learn coping strategies for night-shift work.

Covalent modification of the pyruvate dehydrogenase complex provides an important regulatory mechanism for controlling the disposal of glucose and other compounds metabolized to pyruvate. Regulation of the complex by this mechanism is achieved in part by tissue-specific expression of the genes encoding isoenzymes of pyruvate dehydrogenase kinase (PDK). Starvation is known from our previous work to increase PDK activity of heart and skeletal muscle by increasing the amount of PDK isoenzyme 4 (PDK4) present in these tissues. This study demonstrates that increased expression of both PDK4 and PDK2 occurs in rat liver, kidney, and lactating mammary gland in response to starvation. PDK4 and PDK2 message levels were also increased by starvation in the two tissues examined (liver and kidney), suggesting enhancement of gene transcription. Changes in PDK2 message and protein were of similar magnitude, but changes in PDK4 message were greater than those in PDK4 protein, suggesting regulation at the level of translation. In contrast to these tissues, starvation had little or no effect on PDK2 and PDK4 protein in brain, white adipose tissue, and brown adipose tissue. Nevertheless, PDK4 message levels were significantly increased in brain and white adipose tissue by starvation. The findings of this study indicate that increased expression of PDK isoenzymes is an important mechanism for bringing about inactivation of the pyruvate dehydrogenase complex during starvation in many but not all tissues of the body. The absence of this mechanism preserves the capacity of neuronal tissue to utilize glucose for energy during starvation.

This study investigated whether conditions known to alter the activity and phosphorylation state of the pyruvate dehydrogenase complex have specific effects on the levels of isoenzymes of pyruvate dehydrogenase kinase (PDK) in rat heart. Immunoblot analysis revealed a remarkable increase in the amount of PDK4 in the hearts of rats that had been starved or rendered diabetic with streptozotocin. Re-feeding of starved rats and insulin treatment of diabetic rats very effectively reversed the increase in PDK4 protein and restored PDK enzyme activity to levels of chow-fed control rats. Starvation and diabetes also markedly increased the abundance of PDK4 mRNA, and re-feeding and insulin treatment reduced levels of the message to that of controls. In contrast with the findings for PDK4, little or no changes in the amounts of PDK1 and PDK2 protein and the abundance of their messages occurred in response to starvation and diabetes. The observed shift in the relative abundance of PDK isoenzymes probably explains previous studies of the effects of starvation and diabetes on heart PDK activity. The results indicate that control of the amount of PDK4 is important in long-term regulation of the activity of the pyruvate dehydrogenase complex in rat heart.

Isolated colonocytes have more capacity for the oxidation of isobutyrate and alpha-ketoisovalerate than isolated enterocytes. Both enterocytes and colonocytes express high levels of 3-hydroxyisobutyryl-CoA hydrolase, an enzyme activity important in maintaining low intracellular concentrations of methacrylyl-CoA, a common, potentially toxic intermediate in the catabolic pathways of these compounds. In spite of comparable 3-hydroxyisobutyryl-CoA hydrolase activities in both cell types, and much greater amounts of 3-hydroxyisobutyrate dehydrogenase in colonocytes than in enterocytes, only the colonocytes produced 3-hydroxyisobutyrate as an endproduct of alpha-ketoisovalerate and isobutyrate catabolism. Butyrate very effectively inhibits isobutyrate catabolism by colonocytes, most likely by competitively inhibiting activation of isobutyrate to its CoA ester. Oleate also inhibits isobutyrate catabolism, but at a site more distal than butyrate. Starvation of rats for 72 h decreased the capacity of colonocytes for butyrate but not isobutyrate catabolism. We conclude that isobutyrate could function as a carbon source for energy and anapleurosis in colonocytes under conditions of defective butyrate oxidation or low butyrate availability.

The objective of this study was to assess the essential fatty acid status of patients with anorexia nervosa. Blood was collected from eight fasting female anorexia nervosa patients with a mean of 81% ideal body weight. Fatty acid composition of phospholipids, nonesterified fatty acids, triglycerides and cholesteryl esters of plasma were determined by capillary gas chromatography to indicate polyunsaturated fatty acids status compared with 19 healthy female adults < 25 y old. Subjects with anorexia nervosa showed polyunsaturated fatty acid deficiencies in plasma phospholipids different from simple nutritional essential fatty acid deficiency or chronic malnutrition. The phospholipid profile showed significantly lower (n-6) and (n-3) elongation and desaturation products, and elevated short-chain saturated, short-chain monounsaturated, branched-chain and odd-chain fatty acids. These elevations indicate enhancement of biosynthesis of alternative fatty acids that only partially compensated for the loss of polyunsaturated fatty acids in providing membrane "fluidity." Calculated mean melting point of the fatty acids of phospholipids in patients with anorexia nervosa was elevated 7.7 degrees C above normal values. These results demonstrate that patients with anorexia nervosa have deficiencies of selected essential fatty acids, compensatory changes in nonessential fatty acids and decreased fluidity of plasma lipids.

The branched-chain alpha-ketoacid dehydrogenase complex, catalyst for the rate-limiting step of branched-chain amino acid catabolism, is controlled by a highly specific protein kinase (branched-chain alpha-ketoacid dehydrogenase kinase) that associates tightly with the complex. The activity state (proportion of the enzyme in its active, dephosphorylated state) of the complex varies dramatically in different rat tissues. The activity state of the complex in the liver is greater than that in any other tissue, and liver contains the lowest amount of kinase protein and kinase mRNA. However, protein malnutrition, a condition under which the complex is largely phosphorylated and inactive, resulted in a three- to fourfold increase in hepatic kinase activity with an accompanying increase in amounts of kinase protein and mRNA. Refeeding a 50% protein diet restored the normal activity state and the original levels of kinase protein and mRNA. The amount of kinase protein associated with the complex rather than changes in specific activity of the kinase appears responsible for observed differences in activity states of the complex in several rat tissues tested. Accordingly, the levels of kinase protein and mRNA measured are highest in tissues with greatest kinase activity (heart > kidney > liver), correlating reasonably well inversely with activity state of the branched-chain alpha-ketoacid dehydrogenase complex in the respective tissues. These observations suggest that the amount of kinase protein expressed in various tissues and in response to dietary protein deficiency is an important factor determining the activity state of the complex.

3T3-L1 fibroblasts have limited enzymatic capacity to oxidize valine. Enzymes expressed in these cells allow efficient oxidation of only the first carbon of this branched chain amino acid. The pathway is effectively truncated at the level of 3-hydroxyisobutyrate because of very low expression of two enzymes required for the complete pathway, 3-hydroxyisobutyrate dehydrogenase and methylmalonate semialdehyde dehydrogenase. These two enzymes, as well as the branched chain alpha-ketoacid dehydrogenase, are markedly induced upon differentiation of 3T3-L1 fibroblasts into adipocytes. Flux through the first two decarboxylation steps of valine catabolism is increased dramatically after differentiation, particularly through the step catalyzed by methylmalonate semialdehyde dehydrogenase. Activation of the distal portion of the valine catabolic pathway correlates with significant increases in enzyme protein and mRNA levels for 3-hydroxyisobutyrate dehydrogenase and methylmalonate semialdehyde dehydrogenase, and this establishes the pathway in 3T3-L1 adipocytes for utilization of valine carbon for lipogenesis. The induction profiles of 3-hydroxyisobutyrate dehydrogenase and methylmalonate semialdehyde dehydrogenase are very similar, suggesting coordinate regulation of the expression of these two valine pathway-specific enzymes. Induction of valine catabolism in 3T3-L1 cells is solely differentiation dependent, suggesting regulation by the same factors that govern differentiation of 3T3-L1 fibroblasts into adipocytes.

Objective: Prospective studies were conducted to test the hypothesis that infants unlikely to have serious bacterial infections (SBI) can be accurately identified by low risk criteria., Methods: Febrile infants (rectal T > or = 38 degrees C) < or = 60 days of age were considered at low risk for SBI if they met the following criteria: 1) appear well; 2) were previously healthy; 3) have no focal infection; 4) have WBC count 5.0-15.0 x 10(9) cells/L (5000-15,000/mm3), band form count < or = 1.5 x 10(9) cells/L (< or = 1500/mm3), < or = 10 WBC per high power field on microscopic examination of spun urine sediment, and < or = 5 WBC per high power field on microscopic examination of a stool smear (if diarrhea). The recommended evaluation included the culture of specimens of blood, cerebrospinal fluid, and urine for bacteria. Outcomes were determined. The negative predictive values of the low risk criteria for SBI and bacteremia were calculated., Results: Of 1057 eligible infants, 931 were well appearing, and, of these, 437 met the remaining low risk criteria. Five low risk infants had SBI including two infants with bacteremia. The negative predictive value of the low risk criteria was 98.9% (95% confidence interval, 97.2% to 99.6%) for SBI, and 99.5% (95% confidence interval, 98.2% to 99.9%) for bacteremia., Conclusions: These data confirm the ability of the low risk criteria to identify infants unlikely to have SBI. Infants who meet the low risk criteria can be carefully observed without administering antimicrobial agents.

Levels of expression of two subunits of the liver branched-chain alpha-ketoacid dehydrogenase complex in response to extremes of dietary protein intake (50% versus 0% protein diet) were determined by quantitative immunoblotting. Dietary protein deficiency decreased the amount of E1 alpha protein to a greater extent than E2 protein. The ratio of E1 alpha to E2 was below 1 in the liver of animals starved for protein and above 1 in the liver of animals fed the high-protein diet. Supplementation of the 0% protein diet with 5% leucine (but not 5% valine) had the same effect as the 50% protein diet. The extremes of dietary protein also resulted in a divergent pattern of expression of the mRNAs for the subunits of the complex. The E1 beta message showed the expected corollary of being greater in the liver of the high-protein-fed rats than the no-protein-fed rats. In contrast, the E2 message was not affected by the two extremes of dietary protein and the E1 alpha message was greater in the liver of the no-protein-fed rats than the high-protein-fed rats. Thus, coordinate regulation of gene expression of the subunits of the complex does not occur in response to dietary protein. Post-transcriptional regulatory mechanisms most likely determine the amount of the complex and the ratio of its subunits. The decrease in E1 alpha/E2 protein ratio that occurs in dietary protein deficiency may increase sensitivity of the complex to phosphorylation-mediated inhibition by branched-chain alpha-ketoacid dehydrogenase kinase.

Severe ketotic diabetes induced in rats by streptozotocin resulted in a reduction in activity of the hepatic branched-chain alpha-ketoacid dehydrogenase complex, regardless of whether activity was expressed on the basis of liver wet weight, total liver, liver protein, or liver DNA. A decrease in enzyme specific activity (units of enzyme activity per mg of enzyme protein) was found responsible for the reduction in measurable enzyme activity of the complex. Insulin treatment reversed the decrease in enzyme specific activity. Treatment of tissue extracts with phosphoprotein phosphatase had no effect, indicating that activity of the complex was decreased by some mechanism other than reversible phosphorylation. Specific protein components of the complex were also not found reduced by the diabetic state. Induction of severe ketotic diabetes in rats previously fed a low-protein diet resulted in activation of the enzyme as a consequence of dephosphorylation. Nevertheless, the specific activity of the dephosphorylated enzyme of diabetic, low-protein-fed rats was decreased relative to that of control, low-protein-fed animals. Reconstitution studies with tissue extracts fortified with the purified E1 component indicate that severe diabetes induces a defect in this component of the hepatic branched-chain alpha-ketoacid dehydrogenase complex.

The old dogma of conservative management and hospitalization for all young febrile infants may no longer be necessary or considered the best option for many febrile infants encountered by practitioners today. All infants will continue to require individualized decisions regarding their care by astute clinicians well-versed in the best options for the management of these children. The suggestions outlined in this article are meant to be a guide for the clinician to the available options for the evaluation and management of the febrile infant 60 days of age or younger.

Ethanol inhibited glucose synthesis from alpha-ketoisovalerate by isolated rat hepatocytes without significant inhibition of flux through the branched-chain alpha-ketoacid dehydrogenase complex. Accumulation of 3-hydroxyisobutyrate, an intermediate in the catabolism of alpha-ketoisovalerate, was increased by ethanol, indicating inhibition of flux at the level of 3-hydroxyisobutyrate dehydrogenase. 3-Hydroxybutyrate caused the same effects as ethanol, suggesting inhibition was a consequence of an increase in the mitochondrial NADH/NAD+ ratio. Flux through the 3-hydroxyisobutyrate dehydrogenase was more sensitive to regulation by the mitochondrial NADH/NAD+ ratio than flux through the branched-chain alpha-ketoacid dehydrogenase. Oleate also inhibited glucose synthesis from alpha-ketoisovalerate, but marked inhibition of flux through the branched-chain alpha-ketoacid dehydrogenase complex was caused by this substrate.

The mitochondrial kinases responsible for the phosphorylation and inactivation of rat heart pyruvate dehydrogenase complex and the rat liver and heart branched-chain alpha-ketoacid dehydrogenase complexes have been purified to homogeneity. The branched-chain alpha-ketoacid dehydrogenase kinase is composed of one subunit with a molecular weight of 44 kDa; pyruvate dehydrogenase kinase has two subunits with molecular weights of 48 (alpha) and 45 kDa (beta). Proteolysis maps of branched-chain alpha-ketoacid dehydrogenase kinase and the two subunits of pyruvate dehydrogenase kinase are different, suggesting that all subunits are different entities. The alpha subunit of the rat heart pyruvate dehydrogenase kinase was selectively cleaved by chymotrypsin with concomitant loss of kinase activity, as previously shown for the bovine kidney enzyme, suggesting that the catalytic activity of pyruvate dehydrogenase kinase resides in this subunit. Polyclonal antibodies against branched-chain alpha-ketoacid dehydrogenase kinase, purified by an epitope selection method, bound only to the 44 kDa polypeptide of the branched-chain alpha-ketoacid dehydrogenase complex, substantiating that the 44 kDa protein corresponds to the kinase for this complex. Both kinases exhibited strong substrate specificity toward their respective complexes and would not inactivate heterologous complexes. The kinases possessed slightly different substrate specificities toward histones. Phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase complex by its purified kinase was inhibited by alpha-chloroisocaproate and dichloroacetate, established inhibitors of the phosphorylation of the complex. cDNAs encoding the branched-chain alpha-ketoacid dehydrogenase kinase have been isolated from rat heart and rat liver lambda gt11 libraries. This represents the first successful cloning of a mitochondrial protein kinase. Preliminary data suggest that two different isoforms of the kinase may exist in different ratios in various tissues. No evidence was found for induction of the branched-chain alpha-ketoacid dehydrogenase complex nor its kinase by clofibric acid. Rather, clofibric acid is a potent inhibitor of the activity of the branched-chain alpha-ketoacid dehydrogenase kinase and this may be the molecular mechanism responsible for the myotonic effects of clofibric acid in man.

Unlabelled: A decrease in the myocardial extraction or excretion of lactates indicates its hypoxia. The authors analyzed changes in the extraction of lactates, non-esterified fatty acids (NEFA) and their fractions: C 14:0, C 16:0, C 16:1, C 18:1, C 18:2 in a group of 15 patients with dilated cardiomyopathy (DC) and in 10 controls at rest and after the cold pressor test (CPT). At rest, 5 patients with DC produced lactates or extracted less than 10%. During CPT, production of lactates was observed in another four patients (mean values: 17.9 +/- 32.6% vs 24.5 +/- 5.2%, ns). NEFA extraction was found in the DC group: 18.4 +/- 20.6% vs. 13.9 +/- 33.3% /p < 0.05/). The acids involved were C 14:0, C 16:0, C 16:1 and C 18:0. In the controls, NEFA extraction did not change significantly: 12.1 +/- 8.6% vs 29.7 +/- 33.2%., Conclusions: 1. In part of patients with DC, production of lactates was found. The degree of lactate excretion increased due to CPT; 2. In DC, adrenergic stimulus (CPT) impairs the myocardial utilization of NEFA. This applied mainly to myristic acids, palmitic acid, palmitooleic acid and stearic acid; 3. This phenomenon probably is a result of profound functional and morphological damage to the mitochondrial system in DC.

Previously healthy infants younger than 2 months of age without evidence of soft tissue or musculoskeletal infection who had white blood cell counts between 5000 and 15,000/mm3, band form counts less than or equal to 1500/mm3, urinalysis less than or equal to 10 white blood cells/high power field (spun sediment) and stool less than or equal to 5 white blood cells/high power field (if diarrhea) were considered at low risk for a serious bacterial infection. Infants meeting these criteria whose parents were judged to be adequate observers and had a telephone and automobile were eligible for outpatient management. Infants were given ceftriaxone to cover the possibility that the low risk criteria might miss more infants with serious bacterial infections than was predicted. From Jan. 1, 1987 to May 31, 1989, 86 infants younger than 2 months were enrolled. There were no serious complications in these infants. Twelve had transient problems possibly related to the intramuscular ceftriaxone therapy. One low risk infant was hospitalized for Neisseria meningitidis bacteremia and five other infants were hospitalized for medical or social reasons. All six hospitalized infants had short admissions and did well. This study supports the continued use of the low risk criteria to distinguish infants unlikely to have a serious bacterial infection. Furthermore, in a selected group of low risk infants, outpatient management may be an acceptable alternative to inpatient therapy.