Lactate Dehyrogenase Research Articles

Preservation of the genetic diversity of the sevryuga (Acipenser stellatus Pall) in conditions of artificial reproduction

According to the lactadehydrogenase locus, the genetic diversity of 9 samples of the Volga sevryuga of different origin was studied. In total, 733 pieces of young sevryuga, 99 pieces of producers and 107 pieces were studied. four-year-olds of sevryuga with MPERZ. To assess the genetic variability of lactate dehyrogenase allozymes, the method of polyacrylamide gel electrophoresis was used, as the most appropriate for assessing the influence of the environment in natural and domesticated populations. It is shown that the young sevryuga, grown in the conditions of 6 Astrakhan sturgeon hatcheries, differs significantly from the control group of natural producers. The LDH-3 lactate dehydrogenase locus is represented by 6 genotypes. 5 genotypes were found in producers, in the absence of only genotype 100/112. Genotypes 70/112 and 112/122 are missing in the samples from all plants and MPERZ, while genotype 70/70, which was found in excess at MPERZ, is also missing in three plants. However, in general, only 2 genotypes out of 6 are missing for all ARI. A study of the dynamics of the frequency of alleles in this set of samples showed that in 4 samples with ARI and on MPRZ there is no allele 112. However, in general, the ARI allele persists. Thus, it is shown that due to the different genetic variability of LDH-3 in juveniles from different acute respiratory infections, the genetic diversity in the samples as a whole approaches the indicators of producers. Therefore, the preservation of a variety of growing conditions for young sevryuga will contribute to the preservation of its genetic diversity.

Assessment of serum procalcitonin, adenosine deaminase, lactate dehyrogenase, calcium, phosphorus in tuberculosis patients

Tuberculosis is an infectious disease which is caused by bacteria. The disease is mostly transmitted from person to person, usually by inhaling bacteria – carrying air droplets. Tuberculosis commonly affects the lungs, but it can also affect any other organ of the body such as brain, intestine, kidneys or the spine. Tuberculosis is one of the most ancient disease of mankind for more than four thousand years. It is a chronic disease caused by Mycobacterium tuberculosis and spreads from person to person through air. The new modalities are very much helpful for the diagnosis and treatment of tuberculosis, Unfortunately people are still suffering with this disease. Worldwide it is among the top ten killer infectious disease secondly to HIV and COVID 19. According to World Health Organisation(WHO) tuberculosis is a global pandemic. The present study shows a very strong significant for the assess of serum procalcitonin, adenosine deaminse, lactate dehydrogenase, calcium, phosphorus and thyroid stimulation hormone in tuberculosis patients.

Silver nanoparticles cause osmoregulatory impairment and oxidative stress in Caspian kutum (Rutilus kutum, Kamensky 1901).

Silver nanoparticles (AgNPs) are increasingly used in several industrial and household products because of their antibacterial and antifungal properties. Hence, there is an inevitable risk that these chemicals may end up in aquatic biotopes and have adverse effects on the fauna. In order to assess potential health effects on aquatic organisms, this study evaluated the effects of waterborne AgNP exposure for 7days on a set of critical stress parameters in juvenile Caspian kutum (Rutilus kutum), an economically important fish in the Caspian Sea. The applied level 11μg/l of AgNP is high compared to reported water concentrations and corresponds to 40% of the 96h LC50 value, initially determined to be 28μg/l. Gill heat shock protein 70 (hsp70) mRNA expression, Na+/K+-ATPase activity and enzymatic activities of liver superoxide dismutase (SOD), glutathione peroxidase (Gpx), lactate dehyrogenase (LDH) and alkaline phosphatase (ALP), and whole-body cortisol and thyroid hormones (T3 and T4) were measured as endpoints. Gill hsp70 mRNA expression increased and gill Na+/K+-ATPase activity decreased in AgNP-exposed fish compared to controls. The specific activities of all liver enzymes decreased significantly compared to controls. Whole-body cortisol and thyroid hormones decreased compared to controls. In conclusion, the study demonstrates that AgNPs cause oxidative stress and gill osmoregulatory disruption in Caspian kutum juveniles.

MUSCULAR DYSTROPHIES

Objectives: Muscular dystrophies are classified into different types based ontheir age of onset, clinical severity, rate of progression, distribution of muscles weakness,pattern of inheritance and the genes involved. As muscular dystrophies are relativelyuncommon disorders, very little work has been done in Pakistan. This study has classified (forthe first time in Pakistan) the patients with different types of muscular dystrophies by clinicaland biochemical correlation. Study Design: Observational, descriptive study. Setting:Departments of Morbid Anatomy and Histopathology and Human Genetics and MolecularBiology at University of Health Sciences Lahore. Period: Three years’ (March, 2012-March2015). Methodology: A total of 100 patients comprising of all age groups and both gendersand with strong clinical suspicion of muscular dystrophy, with or without positive family history,were included. Detailed clinical history and physical examination findings were recordedfollowed by estimation of serum Creatine Phosphokinase (CPK), Lactate Dehyrogenase(LDH) and Aldolase levels. Results: A total of 77 males (M) and 23 females (F) were included.Mean age of the patients was 12.1990 + 6.69913 years. Parents of 76 patients reported withconsanguineous marriages while positive family history was reported in 31 patients. Eightytwo cases managed to ambulate with support of other persons while n= 14 patients weretotally non ambulatory. Symmetrical muscle involvement (lower limb followed by upper limb)was shown by 87 cases (including all males and n=22 females). Involvement of the lowerlimbs as a primary weakness followed by symmetrical and severe involvement of the upperlimb girdle and lower limb girdle suggested limb girdle muscle dystrophy (LGMD) in 9 (6F,3M)patients. Involvement of shoulders and upper limbs as a primary weakness followed by facialmuscles and infrequent involvement of lower limbs suggested fascioscapulohumeral muscledystrophy (FSHMD) in 2 cases. Involvement of upper limbs as a primary weakness followedby lower limbs with a moderate rise in CPK levels and mild phenotype was seen in two femalepatient which was quite suggestive of dysferlinopathy. Only two male patients reported onsetof weakness and ambulation loss before 1 year of age that may represent congenital muscledystrophy (CMD), dystroglycanopathy or congenital onset of LGMD (cLGMD). Mean serumCPK, LDH and Aldolase levels were 2376.6364 + 910.78963 U/L, 1030.7800 + 180.1620 U/Land 10.089 + 1.525 U/L respectively that demonstrated significant association (p<0.05) withcharacteristic clinical features. Conclusion: Muscular dystrophies are not only restrictedto dystrophinopathies in our population. Apart from clinical and biochemical parameters,appropriate histological and/or gene mutation analysis is mandatory for precise classificationof these disorders.

The Diagnostic Values of Protein to Lactate Dehyrogenase Ratio in Serum and Pleural Fluid in Exudate Pleural Effusions

Introduction: Different etiologies of pleural effusion are diagnosed based on serum and plural fluid characteristics. The aim of this study was to assess and compare the serum and pleural fluid protein to lactate dehyrogenase (Pr/LDH) ratio in exudative pleural effusions. Materials and Methods: This study was conducted on 60 patients with exudative pleural effusion including: 20 cases with parapneumonic , 20 cases with Tuberculosis (TB), and 20 cases with malignancy. The serum and pleural Pr/LDH were measured and compared among 3 groups. Result: The mean age of the patients was 55±19SD (years) and male to female ratio was 36/24. There was no statistically significant difference in mean age of the patients among 3 groups (p=0.08). There were statistically significant differences in serum and pleural Pr./LDH ratios among groups ( p=0.04 and p= 0.1, respectively). Additionally the comparisons of serum and pleural Pr./LDH ratios between malignancy and tuberculosis groups were significant ( p=0.02 and p=0.001 , respectively). The serum and pleural Pr./LDH ratios were higher in TB group. Conclusion: The results of our study showed that serum and pleural Pr./LDH ratio can be used in differentiating the etiology of exudative pleural effusion, but needs to be confirmed by larger study.

Artifactually elevated serum bicarbonate results caused by elevated serum lactate dehydrogenase concentrations

An accurate estimation of serum bicarbonate concentration (HCO3(-)) is essential to the diagnosis and treatment of acid-base disorders and electrolyte disturbances. We noted significant discrepancies between HCO3(-) concentration measured by the Olympus AU2700 analyser and total serum carbon dioxide (TCO2) concentration derived from a Radiometer blood gas analyser on several patient samples. This was reported to the manufacturer which led to a recall of certain reagent lot numbers. We hypothesised the mechanism for this interference to be elevated levels of lactate dehyrogenase (LD). We investigated the effect of increasing LD concentration on HCO3(-) with the reagent lot that was recalled and compared this with a subsequent reagent lot that was known to be unaffected on an Olympus AU2700. The experimental data confirmed a positive interference in the Olympus AU2700 HCO3(-) assay using older reagent lot numbers. The false positive HCO3(-) interference was significant (>10% change) when the LD concentration exceeded 845 U/L. Very high levels of LD concentrations were the cause of interference in the Olympus AU2700 HCO3(-) enzymatic assay. Laboratorians should be aware that very high levels of LD may be a potential interference in some enzymatic HCO3(-) assays.

Preconditioning of cardiac stem cells by electrical stimulation induces cytoprotection against oxidative stress through release of connective tissue growth factor by miR‐378 downregulation

Survival of stem cells following transplantation in the infarcted myocardium is a critical issue for the cell‐based therapy and preconditioning (PC) of stem cells is a method for cytoprotection. In this study, we investigated the cytoprotective effect of PC with electrical stimulation (EleS) on cardiac stem cells (CSCs) survival. Sca‐1+ CSCs were isolated from male C57BL6 mice hearts. PC of CSCs with EleS (EleSCSCs) was carried out for 3 h at 1.5 V followed by exposure to H2O2. Cytoprotective effects and cell adhesion ability were significantly increased by EleS as evaluated by TUNEL, lactate dehyrogenase release assay and adhesion assay as well as Sry gene analysis in vivo. EleS increased phosphorylation of AKT, FAK and GSK3β, and decreased pro‐apoptotic protein caspase‐3 cleavage. Pretreatment with Wortmannin, a PI3K inhibitor, and FAK inhibitor‐14 abolished the pro‐survival effects of EleS. Importantly, we found that connective tissue growth factor (Ctgf) is a key determinant for EleS‐induced CSC survival and adhesion. Furthermore, we identified miR‐378 is a potential negative regulator for Ctgf expression in EleSCSCs.In conclusion, EleS enhanced CSC survival in vitro and in vivo through AKT/FAK/CTGF/miR‐378 signaling cascade, being suggested as a novel preconditioining strategy.This work was supported by National Institutes of Health grant, [HL087246 to M.A.].

Sulfiredoxin‐1 protects PC12 cells against oxidative stress induced by hydrogen peroxide

Oxidative stress results in protein oxidation and is implicated in cerebral disease, such as Parkinson's disease, Alzheimer's disease, and ischemic stroke. Sulfiredoxin-1 (Srxn1) is an endogenous antioxidant protein that has neuroprotective effects. The mechanisms of Srxn1 in oxidative stress have not been well studied, however. This study used 180 μM H2 O2 exposure for 24 hr to model oxidative stress. This experimental design allowed us to explore the protective effects and underlying mechanisms of Srxn1 in PC12 cells. To investigate Srxn1's role in oxidative stress protection, transient knockdowns of Srxn1 in PC12 cells were performed prior to treatment with 180 μM H2 O2 for 24 hr. Knockdown of Srxn1 resulted in decreased cell viability and increased cellular damage as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and lactate dehyrogenase analysis, respectively. Intracellular superoxide dismutase and glutathione are important indexes of oxidative stress; these were reduced in Srxn1 knockdown PC12. We further found that the decreased Srxn1 correlated with a reduction in 2-Cys Prdxs activity. Moreover, 2-Cys Prdxs protein levels were increased in the H2 O2 -dosed cells, as measured by RT-PCR and immunoblot analysis. These results suggested that Srxn1 can protect PC12 cells from H2 O2 -induced oxidative stress and are involve in Prdxs activity. Srxn1 play a protective role against oxidative injury and demonstrates potential as a target for neuroprotective intervention in oxidative stress.

1146

Case Reports: Disulfiram is an alcohol deterrent used for chronic alcoholism. Fulminant liver failure is a rare but potentially fatal adverse drug reaction with disulfiram. Only 4 cases have been reported in literature who received liver transplant after disulfiram induced fulminant hepatic failure. We report a case of a 42 year old female, with no prior history of liver disease, on disulfiram for 8 months for the treatment of her alcohol dependence. Patient initially presented with 2 days of worsening dull non-radiating abdominal pain. At presentation patient was afebrile, normotensive and saturating 98 % but she was acutely agitated and hallucinating. Bloodwork showed elevated transaminases (AST 18000 IU/L, ALT 5037 IU/L), hyperbilirubinimia and lactic acidosis (18.5 mEq/L). She was intubated for severe metabolic acidosis. Patient was transferred to our tertiary care hospital for management of acute deterioration of mental status, coagulopathy and fulminant hepatic encephalopathy. At presentation patient was intubated, tachycardic and agitated. On examination pupils were 8mm reactive to light bilateral, hepatomeagaly was noticed but tenderness could not be elicited as patient was sedated. Laboratory findings consistent with severe anion gap metabolic acidosis pH 7.16, acute renal failure, ammonia 336 mcg/dl, elevated transaminases, lactate dehyrogenase 7324 U/L, alkaline phosphatase 149 U/L, hyperbilirubinimia, severe coagulopathy. Patient was maintained on supportive care, mechanical ventilation and Continuous Veno-Venous Hemodialysis. After finding no other cause of Liver injury, worsening of encephalopathy and with no signs of improvement with aggressive medical therapy, patient underwent liver transplant for disulfiram induced fulminant liver failure. Post-transplant patient recovered completely from her acute decompensated state. Histopathological analysis of post-transplant liver specimen was consistent with drug induced acute liver toxicity. The authors want to stress the potential importance of monitoring liver function in patients on disulfiram therapy and role of liver transplantation in patients with disulfiram induced fulminant liver failure.

Effective Elimination of CML Progenitor and Stem Cells Through Combination of α-CD123 Antibody-Dependent Cell-Mediated Cytotoxicity and Tyrosine Kinase Inhibitor Treatment

AbstractAbstract 32Since the introduction of tyrosine kinase inhibitor (TKI) therapy overall survival and complete molecular response rates in chronic phase chronic myeloid leukemia (CP-CML) patients have significantly improved. However, leukemic stem cells (LSCs) and progenitor cells persist and are thought to be responsible for disease progression, development of TKI resistance and disease recurrence after stopping TKI therapy. Protection by cytokines, such as IL-3 and GM-CSF, provides a potential mechanism of LSC resistance. While in acute myeloid leukemia (AML) monoclonal antibody (mAb) targeting of IL-3 receptor α (CD123), a recognized marker for AML LSCs, has been studied in vitro and in vivo, similar investigations have not been undertaken in CML to date. CSL362 is a genetically-engineered form of the specific blocking mAb 7G3 optimized for Fc receptor binding to achieve maximal antibody-dependent cell-mediated cytotoxicity (ADCC) capacity. Here we investigate the expression of CD123 in CD34+ progenitors and CD34+CD38− LSCs, isolated from CP- and blast crisis (BC) - CML patients, and study the benefits of targeting those cells by CSL362 alone and in combination with TKIs.Flow cytometry analysis established significantly elevated expression of CD123 on CD34+CD38− cells from CP-CML (53.0 ± 5.8 %, n=16, p=0.003) and BC-CML (73.2 ± 6.7 %, n=18, p<0.001) patients compared to normal donors (20.3 ± 4.2 %, n=8), with clear increases in CD123 expression with disease progression in matched samples (n=2). Subsequent assessment of apoptosis, colony forming unit (CFU-GM) and long-term culture-initiating cell (LTC-IC) potential confirmed the ability of CSL362 to block IL-3-mediated rescue of TKI-induced cell death. However, in the presence of other cytokines, likely found in the physiological bone marrow microenvironment, this effect was lost.We also demonstrate by lactate dehyrogenase release and clonogenic assays that CML CD34+ cell numbers are significantly reduced, in a dose-dependent manner, by CSL362-induced ADCC employing NK cells from healthy donors (42.4 ± 8.1 % lysis, n=3, and CFU-GM decreased to 30 ± 10.8 % of controls, n=5, p=0.003). In keeping with this, flow cytometry analysis revealed specific elimination of CP- and BC-CML CD123+ CD34+CD38− cells (from 42.9 % to 18.6 %, n=5, p=0.004, and from 71 % to 35.3 %, n=3, p=0.044, respectively). Importantly, autologous CML patient NK cells, collected after achievement of major molecular response, also mediate CSL362-dependent cytotoxicity similar to allogeneic healthy donor NK cells as indicated by equivalent numbers of remaining CFUs (28 ± 6.7 % vs. 34.9 ± 3.4 %, n=5, Fig. A). We further have evidence to suggest preferential elimination of CML over normal LTC-ICs (30.3 ± 9.9 % vs. 62.6 ± 11.2 % remaining, n=3, p=0.096) in the autologous setting. Of clinical importance, the combination of Nilotinib and CSL362 resulted in a significantly greater reduction in CFUs (additive effect) when compared to either agent alone (Fig. B).Taken together these data suggest that selective ADCC-mediated lysis, likely the major mode of action of CSL362 in vivo, efficiently eliminates CML progenitor and stem cells. Promising results evaluating CSL362/TKI combination treatments, with the expectation to further enhance specificity for leukemic while sparing normal progenitor and stem cells as indicated from preliminary experiments, warrant further studies. [Display omitted] Disclosures:Nievergall:CSL Ltd: Research Funding. White:BMS: Research Funding; CSL Ltd: Research Funding; Novartis Oncology: Honoraria, Research Funding. Ramshaw:CSL Ltd: Research Funding. Busfield:CSL Ltd: Employment. Vairo:CSL Ltd: Employment. Lopez:CSL Ltd: Research Funding. Hughes:Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Ltd: Research Funding. Hiwase:CSL Ltd: Research Funding.

Experimental Cassia senna intoxication in Lohmann broiler chicks

The aim of this study was to evaluate the toxic effects of Cassia senna fruits in Lohman broiler chicks. Chicks fed with diet containing 2 and 5% C. senna fruit for 4 weeks, thereafter the chicks in the two groups were fed with control diet for 2 weeks. Depression in body weight, weight gain, inefficiency of feed utilization and anaemia was observed in chicks fed with 2 and 5% C. senna compared with the control diet. There were significant increase in the serum values of sorbitol dehydrogenase, lactate dehyrogenase, glutamate dehydrogenase, glutmate oxaloacetate transaminase, acid phosphatase uric acid and significant decrease in the blood values of haemoglobin concentration (Hb), packed cell volume (PCV) and red blood corpuscle (RBC) in test chicks were compared to those of the control group. Liver showed vacuolation of the centrilobular hapatocytes in the two groups. Kidneys showed degeneration and or necrosis of the epithelial cells of the tubules, scattered lymphoid nodules and shrinkage of the glomerular tufts. Catarrhal enteritis and focal degeneration of the intestinal mucosa were observed in test groups. At the end of the period following withdrawal of senna meals the damage to the vital organs was reduced but neither the hepatocytes nor the cells of the renal tubules had completely reverted to normal. Key words: Cassia senna, toxicity, chicks, haematology and biochemistry.

CD5-positive mucosa-associated lymphoid tissue (MALT) lymphoma: a clinicopathologic study of 14 cases

Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) is a B-cell neoplasm that is typically CD5 negative. We describe the clinicopathologic, immunophenotypic, and cytogenetic features of 14 cases of CD5+ MALT lymphoma. There were 9 men and 5 women (median age, 68 years; range, 34-87 years). MALT lymphoma was initially diagnosed in salivary glands (n = 4), nasopharynx (n = 2), and 1 case each in conjunctiva, thyroid, stomach, colon, skin, lung, kidney, and retroperitoneum. Two patients had localized disease; 9 had disseminated disease with generalized lymphadenopathy (n = 8), multifocal lymphoma (n = 6), or bone marrow involvement (n = 5). No staging information was available for the remaining patients. None presented with B symptoms, splenomegaly, cytopenias, lymphocytosis, monoclonal gammopathy, or elevated serum lactate dehyrogenase. Serum β2-microglobulin was elevated in 6. Morphologically, the neoplasms had features typical of MALT lymphoma being composed of small- to medium-sized cells with round to slightly irregular nuclear contours and moderate amount of cytoplasm. Lymphoepithelial lesions were noted in 4 cases. CD5 was positive in all cases by immunohistochemistry (n = 12) and/or flow cytometry (n = 11). All cases assessed were negative for cyclin D1 (13/13) and CD10 (11/11). Conventional cytogenetics in 7 cases showed trisomy 3 in 3 and diploid in 4. With a median follow-up of 71 months (range, 2-131 months), overall survival at 5 years was 100%, although 5 patients required chemotherapy. Our results show that CD5 expression is rare in MALT lymphoma, and is often associated with nongastric disease and an increased tendency to present with disseminated disease. Overall survival is excellent with appropriate therapy.

Blood (1-&gt;3)- -D-Glucan as a Diagnostic Test for HIV-Related Pneumocystis jirovecii Pneumonia

(See the editorial commentary by Morris and Masur, on pages 203-204.) Improved noninvasive diagnostic tests for Pneumocystis jirovecii pneumonia (PCP) are needed. We evaluated the test characteristics of plasma (1 → 3)-β-D-glucan (β-glucan) for HIV-related PCP among a large group of patients presenting with diverse opportunistic infections (OIs). The study population included all 282 participants in AIDS Clinical Trials Group A5164, a study of early versus deferred antiretroviral therapy in conjunction with initial therapy of acute OIs. Baseline plasma samples were assayed for β-glucan, with standard assay reference values defining ≥ 80 pg/mL as positive. Before this analysis, diagnosis of PCP was independently adjudicated by 2 study investigators after reviewing reports from study sites. A total of 252 persons had a β-glucan result that could be analyzed, 173 (69%) of whom had received a diagnosis of PCP. Median β-glucan with PCP was 408 pg/mL (interquartile range [IQR], 209-500 pg/mL), compared with 37 pg/mL (IQR, 31-235 pg/mL) without PCP (P < .001). The sensitivity of β-glucan dichotomized at 80 pg/mL for the diagnosis of PCP was 92% (95% confidence interval [CI], 87%-96%), and the specificity was 65% (95% CI, 53%-75%); positive and negative predictive values were 85% (95% CI, 79%-90%) and 80% (95% CI, 68%-89%) respectively, based on the study prevalence of 69% of patients with PCP. Rates of abnormal lactate dehyrogenase levels did not differ significantly between those with and without PCP. Blood (1 → 3)-β-D-glucan is strongly correlated with HIV-related PCP. In some clinical centers, this may be a more sensitive test than the induced sputum examination and could reduce the need for both bronchoscopy and empirical therapy of PCP.

Cryopreservation of whole ovaries with vascular pedicles: vitrification or conventional freezing?

To compare the efficacy of vitrification and conventional freezing of whole ovaries. Ovaries obtained from 5-year-old female bovines were cryopreserved by conventional freezing, rapid freezing and vitrification. The ovarian cortical strips were cryopreserved by conventional freezing. Follicular viability was assessed using the trypan blue test; the percentage of morphologically normal primordial follicles, hormones concentrations in the culture supernatants, and lactate dehyrogenase levels were measured. The efficacy of cryopreservation of whole ovaries by vitrification was higher than those by conventional freezing and rapid freezing. Conventional freezing of ovarian cortical strips was more effective than cryopreservation of whole ovaries by conventional freezing, rapid freezing, and vitrification. Vitrification seems to be more suitable than conventional freezing for cryopreservation of whole ovaries. However, further studies are required to improve the efficacy of vitrifying whole ovaries.

Prognostic significance of cytochrome C release and tissue lactate dehydrogenase in patients with breast cancer

The aim of this study was to evaluate the prognostic significance of cytochrome c release and tissue lactate dehyrogenase (LDH) in patients with breast cancer. This study included 90 patients with breast cancer and 35 patients with benign breast lesions. In cytosolic fraction, release of cytochrome c from mitochondria to cytosol was detected by Western blot, while total LDH activity was measured spectrophotometrically. Tissue LDHA mRNA was assessed by RTPCR. We observed a significant increase in cytochrome c release, LDH activity and LDHA mRNA in patients with breast cancer compared to patients with benign breast lesions. Cytochrome c release was significantly declined in poorly differentiated tumors (grade 3)compared to well-differentiated and moderately differentiated tumors (grades 1, 2); and in stages III and IV compared to stages I and II ofbreast cancer. Levels of LDH activity and LDHA mRNA were remarkably elevated in breast cancer patients with grade 3 compared to those with grades 1, 2; and in stages III and IV compared to stages I and II of breast cancer. High levels of LDH activity and LDHA mRNA were detected in breast cancer patients with positive lymph node compared to those with negative lymph node. These data indicate that cytochrome c release, cytosolic LDH activity and tissue LDHA mRNA can be considered as useful prognostic markers in patients with breast cancer.Keywords: Breast cancer, cytochrome c release, LDH, prognosticmarkers

Proteomic analysis of metastatic laryngeal squamous cell carcinoma

Objectives. Patients presenting with head and neck cancer may have no clinical or radiological evidence of cervical lymph node metastasis – the N0 neck. Despite this, histo-pathological data has confirmed the presence of occult metastases in up to 30% of N0 necks (i.e. N+), depending on the site of the primary tumour. Currently there is no way of identifying affected individuals and consequently, 70% may be overtreated.1 Using proteomic and immuno-histochemical (IHC) techniques, we attempted to identify a primary tumour phenotype, which would allow the discrimination of laryngeal squamous cell carcinoma (LSCC) with metastatic potential. Method. Six fresh frozen primary LSCC samples; three each from patients with a histopathologically confirmed N+ or N0 neck were used. Following homogenisation of the tumour tissue, protein separation using 2D gel electrophoresis was performed. Differences in individual protein expression between N0 and N+ groups were identified using Progenesis SameSpots® gel analysis software. Differentially expressed proteins were extracted from the gels and identification was achieved using a combination of matrix-assisted laser desorption and ionization mass spectrometry (MS) (MALDI) and liquid chromatography tandem MS (LC-MS/MS). Results. Ten strongly differentially expressed proteins were identified. Those identified included heat shock protein 90, T complex protein 1 gamma, Bromodomain PHD finger protein 3 and lactate dehyrogenase A, which were up-regulated and thymidine phosphorylase which was down-regulated in N+ samples. Conclusions. These proteins represent potential biomarkers of metastatic potential in LSCC. Further investigation of their worth as biomarkers is necessary using, in the first instance, large sample tissue microarrays. Reference. 1 Yarbrough W.G., Slebos R.J.C. & Liebler D. (2006) Proteomics: clinical applications for head and neck squamous cell carcinoma. Head Neck. 28, 549–558

Microarray Analysis of the Overlap Between Genes Known to Be Induced by Hypoxia and/or Hypoxia-inducible Factor 1α (HIF-1α) and Genes Rapidly Induced by Estrogen in Rat Uterine Epithelial Cells.

Estrogen is the master regulator of gene transcription in the uterine endometrium, acting primarily through its α receptor (ERα). Recent work in our laboratory identified hypoxia-inducible factor 1α (HIF-1α) as a transcription factor which, along with ERα, is necessary for 17β-estradiol (E2)-induced vascular endothelial growth factor (VEGF) expression in the uterus. VEGF causes increased microvascular permeability and edema in the endometrium, which is necessary for subsequent cell proliferation, angiogenesis, and implantation. HIF-1α is normally present in the uterus at low levels and is recruited to the VEGF promoter by E2 via activation of the PI3K/Akt pathway. HIF-1α is also known to regulate the transcription of over 50 other genes in various cell types, many of which are also induced by E2 in the uterus, including most of the glycolytic enzymes, Glut-1, erythropoietin, adrenomedulin, and leptin, in addition to VEGF. Therefore, we hypothesize that E2-induced expression of many genes will involve HIF-1α. To better understand the extent of the overlap between E2- and hypoxia/HIF-1-regulated genes, we used a microarray specific for genes known to be induced by hypoxia and/or to be regulated by HIF-1α (SuperArray Rat Hypoxia Signaling Pathway Oligo GEArray) to determine how many are also induced by E2 in the uterus. Immature female rats were treated with E2 for 0, 1, or 4 h, and the luminal epithelial (LE) cells isolated. Total RNA was extracted and amplified. A biotin tag was attached, and the samples were then hybridized to the array. Of the 114 genes represented, 11 were significantly up-regulated by E2 by 1 and/or 4 h. There was little or no hybridization with the majority of the genes, however, including VEGF, which RT-PCR analysis showed was strongly induced by E2 in the same samples. This indicated that the methodology may be too insensitive to detect many of the target genes, even after E2 stimulation. Nevertheless, increases in the expression of several genes known to be induced by E2 in the uterus were detected, including fos, the glycolytic enzymes enolase, aldolase, and lactate dehyrogenase A, and interleukin 6. In addition, several other genes not previously reported to be controlled by E2 in the uterus were significantly induced. Two of these the transcriptional regulators retinoic acid receptor α (RARα) and inhibitor of DNA binding 1 (Id-1) are of particular interest. RARα was the most strongly up-regulated gene (22-fold). This suggests that it may play an important role in the regulation of gene transcription by E2. Inhibitor of DNA binding 1 (Id-1), which was recently shown to play a role in angiogenesis, was up-regulated 5- and 7-fold by 1 and 4 h, respectively. RT-PCR analysis of both E2-treated rat LE cells and human endometrial cancer (ECC-1) cells was done to confirm these results. These findings support our hypothesis that many of the genes that play a role in cellular adaptations to hypoxia are also required for E2-induced endometrial growth. Future studies will determine exactly how ERα and HIF-1α interact to regulate the expression of this cohort of genes. This work was supported by the NIH/NHLBI-sponsored Institutional Training Grant in Cardiac and Vascular Cell Biology (HL72751) and the NICHD U54 Cooperative Centers Program (HD36207).

Excretory-Secretory Products Produced by Paragonimus westermani Differentially Regulate the Nitric Oxide Production and Viability of Microglial Cells

Background:Tissue-invading helminth parasites secrete a large amount of cysteine proteases that may play critical roles in tissue invasion and immune modulation. However, roles of excretory-secretory products (ESP) secreted by Paragonimus westermani in the activation and death of microglial cells in brain are poorly understood. Objectives: In the present study, we investigated whether ESP could regulate microglial nitric oxide (NO) production and viability. Methods: The NO production and cell viability were assessed by respectively measuring the formation of nitrite and the release of lactate dehyrogenase. Results:At a low (0.2 µg/ml) concentration, ESP significantly stimulated NO production with no apparent cell injury or death in cultured microglial cells. However, at high (≧2 µg/ml) concentrations, ESP induced severe cell death. Inhibition of inducible NO synthase significantly reduced the NO productivity, but not cytotoxicity, of ESP. Similarly, inhibitors of the extracellular signal-regulated kinase, p38 and nuclear factor kappa B also blocked only the NO productivity of ESP. Interestingly, heat inactivation did not hamper the ability of ESP to stimulate microglial NO production. Similarly, pretreatment with thiol-crosslinking reagents dramatically reduced both proteolytic activity and cytotoxicity of ESP, but did not alter NO production in microglial cells. Interestingly, although cysteine protease competitive inhibitors and thiol-alkylating reagents markedly reduced the proteolytic activity of ESP, they did not influence the NO productivity and cytotoxicity of ESP. Conclusion: The present results indicate that the NO production and cytotoxicity by ESP may be differentially regulated via unknown mechanisms, not related with cysteine protease activity.

Aprotinin ameliorates ischemia/reperfusion injury in a rat hind limb model

The aim of the study was to investigate the protective effect of aprotinin in a rat hind limb ischemia/reperfusion (I/R) model. A well-known antioxidant, α-tocopherol, was also tested for comparison. Ischemia was induced for 4 h by vascular clamping of the iliac arteries of 24 Sprague–Dawley rats, followed by 1 h of reperfusion. Muscle injury was evaluated in three groups: a saline group, an α-tocopherol group and an aprotinin group. Blood pH, pO 2, pCO 2, HCO 3, creatine kinase (CPK), lactate dehyrogenase (LDH) and thiobarbituric acid reactive substances (TBARS) as well as muscle TBARS were measured at the end of the reperfusion. Muscle tissue samples were taken for histological examination. α-Tocopherol and aprotinin groups showed a significant amelioration of plasma CPK ( p=0.002, p=0.002), LDH ( p=0.004, p=0.004) and muscle tissue TBARS ( p=0.001, p=0.001) compared with the control. Plasma TBARS were significantly lower in the aprotinin group compared with the control ( p=0.017). Also, tissue TBARS was significantly lower in the aprotinin group than the α-tocopherol group ( p<0.001). Neutrophil infiltration was less prominent in the α-tocopherol and aprotinin groups compared to the control ( p=0.006, p=0.001). These results suggest that aprotinin, a potent anti-inflammatory drug, is more useful than α-tocopherol, a powerful antioxidant, for attenuating muscle injury after I/R.

Linoleic Acid Prevents Chloride Influx and Cellular Lysis in Rabbit Renal Proximal Tubules Exposed to Mitochondrial Toxicants

Despite many studies elucidating the mechanisms of necrotic cell death, the role of fatty acids released during necrosis remains to be determined. The goals of this study were to determine whether linoleic acid could protect rabbit renal proximal tubules (RPT) from necrotic cell death associated with mitochondrial dysfunction and oxidative injury and to determine the mechanisms involved. Exposure to antimycin A (10 μM) for 1 h or hypoxia (perfusion with 95% N2/5% CO2) for 1 or 2 h induced approximately 70% cellular lysis, as measured by lactate dehyrogenase release, versus 10% in controls. Preincubation with linoleic acid (100 μM) fully protected RPT from cellular lysis. RPT were also protected from lysis if linoleic acid was added 15 min after the addition of antimycin A. Measurements of free intracellular Ca2+ concentrations showed that linoleic acid did not prevent the rise in intracellular Ca2+ associated with a 30-min exposure to antimycin A. However, the influx of extracellular 36Cl− following a 30-min exposure to antimycin A was ameliorated in the presence of linoleic acid. Linoleic acid did not prevent cellular lysis after exposure to hypoxia/reoxygenation (1 h/1 h) or t-butyl hydroperoxide (500 μM, 3 h). These data suggest that linoleic acid protects RPT during the late phase of cell death associated with inhibition of the electron transport chain but not oxidative injury. Several other fatty acids also protected RPT from lysis, and structure–activity relationship studies suggest that a free carboxyl terminus and at least one double bond are required for this action.