ABSTRACT
PURPOSE: MKNR3 is a paternally expressed gene whose mutations are the main cause of central precocious puberty (CPP). Protein circulating levels can be easily measured, as demonstrated in idiopathic CPP and healthy controls. No data are available for patients harboring an MKRN3 mutation. Our aim was to perform MKRN3 mutation screening and to investigate if circulating protein levels could be a screening tool to identify MKRN3 mutation in CPP patients. METHODS: We enrolled 140 CPP girls and performed MKRN3 mutation analysis. Patients were stratified into two groups: idiopathic CPP (iCPP) and MKRN3 mutation-related CPP (MKRN3-CPP). Clinical characteristics were collected. Serum MKRN3 values were measured by a commercially available ELISA assay kit in MKRN3-CPP and a subgroup of 15 iCPP patients. RESULTS: We identified 5 patients with MKRN3 mutations: one was a novel mutation (p.Gln352Arg) while the others were previously reported (p.Arg328Cys, p.Arg345Cys, p.Pro160Cysfs*14, p.Cys410Ter). There was a significant difference in circulating MKRN3 values in MKRN3-CPP compared to iCPP (p < 0.001). In MKRN3-CPP, the subject harboring Pro160Cysfs*14 presented undetectable levels. Subjects carrying the missense mutations p.Arg328Cys and p.Gln352Arg showed divergent circulating protein levels, respectively 40.56 pg/mL and undetectable. The patient with the non-sense mutation reported low but measurable MKRN3 levels (12.72 pg/mL). CONCLUSIONS: MKRN3 defect in patients with CPP cannot be predicted by MKRN3 circulating levels, although those patients presented lower protein levels than iCPP. Due to the great inter-individual variability of the assay and the lack of reference values, no precise cut-off can be identified to suspect MKRN3 defect.
Subject(s)
Mutation , Puberty, Precocious , Ubiquitin-Protein Ligases , Humans , Puberty, Precocious/genetics , Puberty, Precocious/blood , Puberty, Precocious/diagnosis , Female , Ubiquitin-Protein Ligases/genetics , Child , Ribonucleoproteins/genetics , Ribonucleoproteins/blood , Child, Preschool , DNA Mutational Analysis , Case-Control Studies , Biomarkers/bloodABSTRACT
The mechanical response of a contractile cell anchored to the substrate through focal adhesions is studied by means of an asymmetric pre-strained tensegrity structure obeying a neo-Hookean stress-strain law. The aim is to assess the influence of overall asymmetric contraction on the cell durotaxis and on the growth of the focal adhesion plaque. The asymmetric kinematics of the system is obtained in two ways, that is by assuming a gradient of the substrate stiffness and through asymmetric buckling. Equivalent springs are purposely considered to represent the stiffness of the ensemble formed by the substrate, the focal adhesion plaque and the integrin ligands. Then, contraction results from elastic strains induced by competing polymerization and actomyosin contraction. The cell mechanical response in terms of durotaxis and its coupling with focal adhesion plaque growth is finally analysed with respect to the effects of asymmetry, gaining some insights into how this asymmetry could participate to redirect cell migration, both in terms of durotaxis and mollitaxis.
Subject(s)
Actin Cytoskeleton , Focal Adhesions , Cell Adhesion/physiology , Focal Adhesions/metabolism , Biomechanical Phenomena , Cell MovementABSTRACT
PURPOSE: We aimed to investigate a cohort of female and male patients with idiopathic central precocious puberty (CPP), negative for Makorin Ring Finger Protein 3 (MKRN3) defect, by molecular screening for Delta-like 1 homolog (DLK1) defects. DLK1 is an imprinted gene, whose mutations have been described as a rare cause of CPP in girls and adult women with precocious menarche, obesity and metabolic derangement. METHODS: We enrolled 14 girls with familial CPP and 13 boys with familial or sporadic CPP from multiple academic hospital centers. Gene sequencing of DLK1 gene was performed. Circulating levels of DLK1 were measured and clinical and biochemical characteristics were described in those with DLK1 defects. RESULTS: A novel heterozygous mutation in DLK1, c.288_289insC (p.Cys97Leufs*16), was identified in a male proband, his sister and their father. Age at onset of puberty was in line with previous reports in the girl and 8 years in the boy. The father with untreated CPP showed short stature. No metabolic derangement was present in the father except hypercholesterolemia. Undetectable Dlk1 serum levels indicated the complete lack of protein production in the three affected patients. CONCLUSION: A DLK1 defect has been identified for the first time in a boy, underscoring the importance of genetic testing in males with idiopathic or sporadic CPP. The short stature reported by his untreated father suggests the need for timely diagnosis and treatment of subjects with DLK1 defects.
Subject(s)
Dwarfism , Sexual Maturation , Male , Female , Humans , Ubiquitin-Protein Ligases/genetics , Mutation , Membrane Proteins/genetics , Phenotype , Calcium-Binding Proteins/geneticsABSTRACT
We demonstrate that several key aspects of the contractile activity of a cell interacting with the substrate can be captured by means of a non linear elastic tensegrity mechanical system made of a tensile element in parallel with a buckling-prone component, and exchanging forces with the surroundings through an extracellular matrix-focal adhesion complex. Mechanosensitivity of the focal adhesion plaque is triggered by pre-strain-driven buckling of the system induced either by pre-contraction or pre-polymerization of the constituents. The impact of pre-polymerization on the mechanical force and the implications of using linear and nonlinear elasticity for the focal adhesion plaque are assessed.
Subject(s)
Focal Adhesions , Mechanotransduction, Cellular , Cell Adhesion , Elasticity , Models, BiologicalABSTRACT
CONTEXT: Hypogonadism in Prader-Willi syndrome (PWS) is generally attributed to hypothalamic dysfunction or to primary gonadal defect. MKRN3, a maternal imprinted gene located on 15q11.2-q13 region, encodes makorin ring finger protein 3, whose deficiency causes precocious puberty, an extremely rare symptom in PWS. OBJECTIVE: This study aimed to evaluate MKRN3 levels in patients with PWS and to analyze its correlation with sexual hormone levels, insulin resistance and Body Mass Index (BMI). METHODS: We performed an observational cross-sectional study and enrolled 80 patients with genetically confirmed diagnosis of PWS with median age of 9.6 years. RESULTS: MKRN3 levels were measurable in 49 PWS patients with a geometric mean of 34.9 ± 22 pg/ml (median: 28.4). Unmeasurable levels of MKRN3 were found in 31 patients. No statistically significant differences were found between patients with and without measurable MKRN3 levels for any clinical, biochemical, or genetic characteristics. However, MKRN3 levels were inversely correlated with HOMA-IR index (p: 0.005) and HbA1c (p: 0.046) values. No statistically significant correlations were found between MKRN3 and LH, estradiol and testosterone concentrations, pubertal development and genetic defect, whereas a direct correlation with FSH was found (p: 0.007). CONCLUSIONS: The typical genetic defect of PWS should lead to unmeasurable levels of the MKRN3 protein due to the inactivation of the paternal allele. Measurable circulating MKRN3 could suggest the possible involvement of tissue-specific imprinting mechanisms and other regulatory factors in gene expression. Correlations with HOMA-IR index, HbA1c, and FSH suggest peripheral actions of MKRN3, but future studies are warranted to investigate this topic.
Subject(s)
Prader-Willi Syndrome , Child , Cross-Sectional Studies , Estradiol , Follicle Stimulating Hormone , Glycated Hemoglobin , Humans , Pilot Projects , Prader-Willi Syndrome/genetics , Testosterone , Ubiquitin-Protein Ligases/geneticsABSTRACT
Building up and maintenance of cytoskeletal structure in living cells are force-dependent processes involving a dynamic chain of polymerization and depolymerization events, which are also at the basis of cells' remodelling and locomotion. All these phenomena develop by establishing cell-matrix interfaces made of protein complexes, known as focal adhesions, which govern mechanosensing and mechanotransduction mechanisms mediated by stress transmission between cell interior and external environment. Within this framework, by starting from a work by Cao et al. (Biophys J 109:1807-1817, 2015), we here investigate the role played by actomyosin contractility of stress fibres in nucleation, growth and disassembling of focal adhesions. In particular, we propose a tensegrity model of an adherent cell incorporating nonlinear elasticity and unstable behaviours, which provides a new kinematical interpretation of cellular contractile forces and describes how stress fibres, microtubules and adhesion plaques interact mechanobiologically. The results confirm some experimental evidences and suggest how the actomyosin contraction level could be exploited by cells to actively control their adhesion, eventually triggering cytoskeleton reconfigurations and migration processes observed in both physiological conditions and diseases.
Subject(s)
Actomyosin , Focal Adhesions , Actomyosin/metabolism , Cell Adhesion/physiology , Focal Adhesions/metabolism , Mechanotransduction, Cellular , Microtubules/metabolismABSTRACT
In recent years, a great deal of research has relied on hypothetical sacrificial dilemmas to investigate decision-making processes involved in pro-social utilitarian choices. Recent evidence, however, has suggested that moral sacrificial choices may actually reflect reduced harm aversion and antisocial dispositions rather than an utilitarian inclination. Here, we used moral dilemmas to confront healthy volunteers with controversial action choices. We measured impulsiveness and venturesomeness personality traits, which have been shown to influence harm aversion, to test their role in utilitarian action and evaluation of moral acceptability. The results of the present study show that, in males, venturesomeness drives engagement in actions and increases moral acceptability. In contrast, in females no effects of venturesomeness were observed on moral action and evaluation. Rather, in females empathetic concern and personal distress, elicited by the vicarious experience of the other's emotional states, exerted an inhibitory effect on action. Taken together, these findings indicate that the "harm aversion hypothesis" may contribute to explain utilitarian choices in males but not in females. In both genders, no association was observed between impulsiveness and moral action.
Subject(s)
Decision Making , Ethical Theory , Harm Reduction , Impulsive Behavior , Men/psychology , Morals , Risk-Taking , Social Behavior , Women/psychology , Adolescent , Adult , Affect , Choice Behavior , Empathy , Female , Humans , Male , Personality , Sex Factors , Young AdultABSTRACT
BACKGROUND: Endothelial function in psoriatic patients has been mainly evaluated through a high-resolution ultrasound measurement of flow-mediated vasodilation in the brachial artery, which is an operator-dependent and technically demanding technique: this characteristic, together with different patient selection criteria, could account for the conflicting results emerging from different studies. Recently, Circulating Endothelial Cells (CECs) level has been suggested as a novel biomarker of vascular injury. METHODS: The number of CECs was determined by a semi-automated immunomagnetic system (CellSearch system) in peripheral blood of psoriatic patients (n = 48) and healthy subjects (n = 50). In 15 patients, CEC level was also evaluated after 6 months of treatment with an anti-TNF-alpha agent, Etanercept. The plasma levels of high-sensitivity C-reactive Protein (CRP), E-selectin, VEGF and PAI-1 were measured by ELISA. The psoriasis severity was assessed by PASI score. RESULTS: A statistically significant difference (P = 0.001) was found between CEC level in psoriatic patients (10.6 ± 9.4 cells/mL) vs. the control group (3.9 ± 0.9 cells/mL). This count inversely correlated with sE-selectin levels (r(2) = 0.16; P = 0.03). After 6 months of therapy, patients experienced a significant (P < 0.05) decrease in CEC levels (3.4 ± 1.3 cells/mL) and in PASI score (from 11.7 ± 8.1 to 2.1 ± 4.0). CONCLUSIONS: The elevated CECs level that we found in a sample of high selected psoriatic patients could be expression of endothelial damage. Lowering of CECs count after treatment with Etanercept support the hypothesis that an effective systemic therapy of psoriasis may also improve the endothelial function.
Subject(s)
Endothelial Cells , Immunoglobulin G/therapeutic use , Psoriasis/blood , Receptors, Tumor Necrosis Factor/therapeutic use , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Adult , Case-Control Studies , Etanercept , Female , Humans , Immunoglobulin G/pharmacology , Immunomagnetic Separation , Male , Middle Aged , Psoriasis/drug therapyABSTRACT
Environmental monitoring is recognized as an important strategy for controlling Listeria monocytogenes in food processing facilities. Samples are taken by swabbing environmental surfaces, and the swabs are immersed in a medium for transport to the laboratory. In this study, buffered peptone water (BPW), Dey-Engley neutralizing broth (DE), neutralizing buffer (NB), Letheen broth (LE), and newly described MCC buffer (MCC) were evaluated as transport media for recovery of sanitizer-stressed L. monocytogenes from inoculated swabs. After storage at 4°C, the media performed similarly, but at 25°C relative recovery efficiency from the inoculated sponges was DE > LE > BPW > MCC > NB. Recoveries from stainless steel surfaces followed similar trends. MCC, DE, and NB were compared for L. monocytogenes recovery in the presence of Escherichia coli, Enterococcus faecalis, Lactobacillus plantarum, Pseudomonas fluorescens, and Listeria innocua. After 4°C storage, all population levels changed little; after 25°C storage, DE allowed the best growth of L. monocytogenes regardless of other species present. MCC, DE, and NB performed similarly for recovery of L. monocytogenes from an artificial milk biofilm and for recovery of Listeria spp. from swabs obtained from a meat processing facility. Transport medium formulation, time and temperature of swab storage, and coexistence of other species affect recovery of sanitizer-stressed L. monocytogenes from environmental swabs. The study confirms the need to maintain 4°C storage conditions during swab transport.
Subject(s)
Environmental Microbiology , Food Contamination/prevention & control , Food-Processing Industry/standards , Listeria monocytogenes/isolation & purification , Colony Count, Microbial , Equipment Contamination , Food Microbiology , Stainless Steel , TemperatureABSTRACT
An ImageJ JavaScript, AUTOCOUNTER, was specifically developed to monitor and measure LC3B-GFP expression in living human astrocytoma cells, namely T98G and U373-MG. Discrete intracellular GFP fluorescent spots derived from transduction of a Baculovirus replication-defective vector (BacMam LC3B-GFP), followed by microscope examinations at different times. After viral transgene expression, autophagy was induced by Rapamycin administration and assayed in ph-p70S6K/p70S6K and LC3B immunoblotting expression as well as by electron microscopy examinations. A mutated transgene, defective in LC3B lipidation, was employed as a negative control to further exclude fluorescent dots derived from protein intracellular aggregation. The ImageJ JavaScript was then employed to evaluate and score the dynamics changes of the number and area of LC3B-GFP puncta per cell in time course assays and in complex microscope examinations. In conclusion, AUTOCOUNTER enabled to quantify LC3B-GFP expression and to monitor dynamics changes in number and shapes of autophagosomal-like vesicles: it might therefore represent a suitable algorithmic tool for in vitro autophagy modulation studies.
Subject(s)
Astrocytoma/physiopathology , Autophagy/physiology , Gene Expression Profiling/methods , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Software/standards , Antibiotics, Antineoplastic/pharmacology , Astrocytoma/genetics , Automation , Autophagy/drug effects , Cell Line , Cell Line, Tumor , Computers , Gene Expression Regulation , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Microscopy, Electron, Transmission , Sirolimus/pharmacologyABSTRACT
AIM: The benefit of coarctation repair on the resolution of systolic hypertension in adults has been questioned. METHODS: Between March 1997 and July 2009, 65 consecutive adult patients (≥ 16 years) underwent repair of aortic coarctation. There were 40 men (65%) and 25 women (35%) with a mean age of 22.3 ± 4.8 years (range, 16 to 34 years). All patients had critical systolic blood hypertension (SBP ≥ 140 mmHg). SBP ranged from 140 to 205 mmHg, with a mean of 163.5 ± 17.6 mmHg. The mean diastolic BP was 95.1 ± 18.3 mmHg (range, 70 to 120 mmHg). Most patients (41/65, 74%) were on a regimen of at least one antihypertensive drug. RESULTS: The patients were followed up after coarctation repair for 2 to 144 months (mean, 68 ± 39 months). There was no death. No other major complications occurred. There have been no repeat interventions during follow-up. Four patients were lost to follow-up. Of the 61 patients with preoperative hypertension, 53 (87%) were normotensive (SBP <140 mmHg) at the most recent follow-up visit. The remaining eight patients showed substantial improvement versus the preoperative status. The mean SBP after operation was 122.5 ± 12.4 mmHg. Mean diastolic blood pressure was 79.5 ± 11.6 mmHg. Forty-one (67%) patients were taking no medication at the last follow-up. CONCLUSION: Surgical repair of coarctation of the aorta in adults can lead to regression of systolic hypertension and a decreased requirement for antihypertensive medication.
Subject(s)
Aortic Coarctation/surgery , Blood Pressure , Hypertension/etiology , Vascular Surgical Procedures , Adolescent , Adult , Antihypertensive Agents/therapeutic use , Aortic Coarctation/complications , Aortic Coarctation/physiopathology , Blood Pressure/drug effects , Female , Humans , Hypertension/drug therapy , Hypertension/physiopathology , Male , Time Factors , Treatment Outcome , Young AdultABSTRACT
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Increased expression of 5-lipoxygenase (5-LO), a key enzyme in the biosynthesis of leukotrienes (LTs), has been reported in MS lesions and LT levels are elevated in the cerebrospinal fluid of MS patients. To determine whether pharmacological inhibition of 5-LO attenuates demyelination, MK886, a 5-LO inhibitor, was given to mice fed with cuprizone. Gene and protein expression of 5-LO were increased at the peak of cuprizone-induced demyelination. Although MK886 did not attenuate cuprizone-induced demyelination in the corpus callosum or in the cortex, it attenuated cuprizone-induced axonal damage and motor deficits and reduced microglial activation and IL-6 production. These data suggest that during cuprizone-induced demyelination, the 5-LO pathway contributes to microglial activation and neuroinflammation and to axonal damage resulting in motor dysfunction. Thus, 5-LO inhibition may be a useful therapeutic treatment in demyelinating diseases of the CNS.
Subject(s)
Arachidonate 5-Lipoxygenase/metabolism , Ataxia/prevention & control , Cuprizone/toxicity , Lipoxygenase Inhibitors/therapeutic use , Multiple Sclerosis/drug therapy , Neuritis/prevention & control , Neurons/drug effects , Animals , Arachidonate 5-Lipoxygenase/genetics , Biomarkers/metabolism , Cerebral Cortex/drug effects , Cerebral Cortex/immunology , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Corpus Callosum/drug effects , Corpus Callosum/immunology , Corpus Callosum/metabolism , Corpus Callosum/pathology , Demyelinating Diseases/chemically induced , Gene Expression Regulation, Enzymologic/drug effects , Indoles/therapeutic use , Interleukin-6/metabolism , Male , Mice , Mice, Inbred C57BL , Microglia/drug effects , Microglia/immunology , Microglia/metabolism , Microglia/pathology , Multiple Sclerosis/metabolism , Multiple Sclerosis/pathology , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/immunology , Neurons/metabolism , Neurons/pathology , RNA, Messenger/metabolismABSTRACT
Phospholipases A(2) (PLA(2)) are the enzymatic keys for the activation of the arachidonic acid (AA) cascade and the subsequent synthesis of pro-inflammatory prostanoids (prostaglandins and tromboxanes). Prostanoids play critical roles in the initiation and modulation of inflammation and their levels have been reported increased in several neurological and neurodegenerative disorders, including multiple sclerosis (MS). Here, we aimed to determine whether brain expression PLA(2) enzymes and the terminal prostagland in levels are changed during cuprizone-induced demyelination and in the subsequent remyelination phase. Mice were given the neurotoxicant cuprizone through the diet for six weeks to induce brain demyelination. Then, cuprizone was withdrawn and mice were returned to a normal diet for 6 weeks to allow spontaneous remyelination. We found that after 4-6 weeks of cuprizone, sPLA(2)(V) and cPLA(2), but not iPLA(2)(VI), gene expression was upregulated in the cortex, concomitant with an increase in the expression of astrocyte and microglia markers. Cyclooxygenase (COX)-2 gene expression was consistently upregulated during all the demyelination period, whereas COX-1 sporadically increased only at week 5 of cuprizone exposure. However, we found that at the protein level only sPLA(2)(V) and COX-1 were elevated during demyelination, with COX-1 selectively expressed by activated and infiltrated microglia/macrophages and astrocytes. Levels of PGE(2), PGD(2), PGI(2) and TXB(2) were also increased during demyelination. During remyelination, none of the PLA(2) isoforms was significantly changed, whereas COX-1 and -2 were sporadically upregulated only at the gene expression level. PGE(2), PGI(2) and PGD(2) levels returned to normal, whereas TXB(2) was still upregulated after 3 weeks of cuprizone withdrawal. Our study characterizes for the first time time-dependent changes in the AA metabolic pathway during cuprizone-induced demyelination and the subsequent remyelination and suggests that sPLA(2)(V) is the major isoform contributing to AA release.
Subject(s)
Arachidonic Acids/metabolism , Cerebral Cortex/metabolism , Cuprizone/toxicity , Demyelinating Diseases/metabolism , Group V Phospholipases A2/metabolism , Myelin Sheath/metabolism , Nerve Tissue Proteins/metabolism , Animals , Astrocytes/drug effects , Astrocytes/immunology , Astrocytes/metabolism , Astrocytes/pathology , Cerebral Cortex/drug effects , Cerebral Cortex/immunology , Cerebral Cortex/pathology , Cyclooxygenase 1/genetics , Cyclooxygenase 1/metabolism , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Demyelinating Diseases/chemically induced , Demyelinating Diseases/immunology , Gene Expression Regulation, Enzymologic/drug effects , Group V Phospholipases A2/genetics , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Macrophages/pathology , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Microglia/drug effects , Microglia/immunology , Microglia/metabolism , Microglia/pathology , Multiple Sclerosis/immunology , Multiple Sclerosis/metabolism , Multiple Sclerosis/pathology , Myelin Sheath/drug effects , Myelin Sheath/immunology , Neurons/drug effects , Neurons/immunology , Neurons/metabolism , Phospholipases A2, Cytosolic/genetics , Phospholipases A2, Cytosolic/metabolism , RNA, Messenger/metabolism , Time FactorsABSTRACT
Several epidemiologic studies have highlighted that latex sensitization prevalence rate has increased over twenty years both in the general and working population, mainly among health care workers. Such subjects can develop immediate local or systemic hypersensitivity reactions up to anaphylactic shock. First, at San Paolo Hospital in Milan, it has been determined latex sensitization and allergy prevalence rates in health care workers involved in health surveillance. Subsequently an interdisciplinary task group has been constituted in order to plan latex pathology preventive actions in health workers and to identify the preventive measures that must be applied in allergic patients. These facets are tightly one another linked. Since latex allergy primary prevention is the most effective, but difficult to put into effect. Operational protocols, by which recognize latex allergy risk factors and reduce exposure to this substance, have developed for both workers and users. Latex allergy and sensitization occurrence will not probably be erased by these procedures application, but they will be reduced within the limits as low as possible.
Subject(s)
Latex Hypersensitivity/prevention & control , Hospitals , Humans , Italy , Risk FactorsABSTRACT
The growth response of Salmonella Enteritidis (SE) on the vitelline membrane in vitro was studied with the use of a special tube devised specifically for the inoculation of SE onto the vitelline membrane and for the sampling of the yolk near the inoculation site. This latter ability allowed the detection of the movement of SE into the yolk. The growth of SE on the membrane was compared with that of SE inoculated into yolk and albumen in vitro and in ovo in fresh in-shell eggs. The incubation time was 2 days, and the incubation temperatures were 4, 8, 15, 27, and 37 degrees C. Comparison of the results obtained for in vitro growth showed that at 4, 8, and 15 degrees C, SE behaved as if it were in the albumen, with its numbers decreasing over time. At 27 and 37 degrees C, SE grew as if it were in yolk, with a maximum increase of 4.5 log CFU after 2 days at 37 degrees C. In no experiments involving growth on the vitelline membrane did SE appear in the yolk. Comparisons between in vitro and in ovo growth responses of SE in yolk and albumen indicate that SE growth on the membrane parallels that in the in-shell egg.
Subject(s)
Eggs/microbiology , Food Microbiology , Salmonella enteritidis/growth & development , Temperature , Animals , Chickens , Colony Count, Microbial , Egg White/microbiology , Egg Yolk/microbiology , Time FactorsABSTRACT
In this study we report the effects of sulfated polysaccharides on the ecto-ATPase activity of intact cells of Leishmania tropica. Increasing concentrations of dextran sulfate stimulated progressively the ecto-ATPase activity, but did not modify other ecto-enzymes present on the surface of this parasite, such as 5'nucleotidase, 3'nucleotidase and a membrane-bound acid phosphatase activity. This stimulation was not observed when other sulfated polysaccharides such as chondroitin sulfates and heparin were tested. It depends on size and charge of the dextran sulfated molecule. When the cells were incubated in the presence of dextran sulfate Mr 8,000; 40,000 and 500,000 the stimulation of the ecto-ATPase activity was 11%; 23%; and 63%, respectively, and the stimulation was not observed when desulfated dextran (Mr 40,000) was used. The effects of dextran sulfate also depend on pH of the medium. At pH 7.5, the stimulation was over 60%, whereas at pH 8.5 only 25%. The effects of dextran sulfate 500,000 on the ecto-ATPase activity was totally abolished by spermidine and partially by putrescine, two polyamines synthesized and released by Leishmania.
Subject(s)
Adenosine Triphosphatases/metabolism , Dextran Sulfate/pharmacology , Leishmania tropica/enzymology , 4-Nitrophenylphosphatase/metabolism , Animals , Apyrase/metabolism , Chondroitin Sulfates/pharmacology , Culture Media , KineticsABSTRACT
The present study examined the prevalence of Salmonella spp. and the prevalence and quantity of generic (biotype I) Escherichia coli on carcasses or in pig feces at a pork processing plant operating under the hazard analysis and critical control point-based inspection models project (HIMP) program. The surfaces of carcasses were sponged on 10 separate days over a 30-day period at two processing steps: (i) immediately following exsanguination (100 carcasses), and (ii) after the carcasses were washed, eviscerated, and chilled overnight (122 carcasses). Feces were also collected from 60 of the 100 sponged, postexsanguinated pigs. Salmonella spp. were detected on 73.0% of the 100 postexsanguinated pigs, in 33.3% of the 60 fecal samples, and on 0.7% of the 122 chilled carcasses. E. coli was found on 100.0% of the postexsanguinated pigs and on 30.1% of chilled carcasses tested. The mean concentration of E. coli on carcasses was 1,700 CFU/cm2 immediately after the exsanguination step and 1.1 CFU/cm2 at the chilled carcass stage. Previous studies at this processing plant showed that the pre-HIMP baseline level of Salmonella spp. on the chilled carcasses was 0.8%, indicating that the present HIMP inspection system produced an equivalent level of bacteriological performance.
Subject(s)
Escherichia coli/isolation & purification , Food Contamination/analysis , Salmonella/isolation & purification , Swine/microbiology , Animals , Colony Count, Microbial , Feces/microbiology , Food Microbiology , Food-Processing Industry , Meat/microbiology , Prevalence , Quality ControlABSTRACT
To develop a hazard analysis and critical control point plan for food processing operations, critical control points must be determined. Swine slaughtering and dressing operations were investigated to establish their critical control points. We monitored the microbiology of swine carcasses by surface swabbing carcass bellies at various steps during the process and by quantitating total aerobic plate count (APC) and coliforms. Starting with a dehaired carcass, the sequential steps monitored included presingeing, postsingeing, polishing, and chilling. Initial results indicate that singeing and chilling substantially reduced the levels of APC and coliforms, whereas polishing increased their levels. The hygienic characteristics of individual operations involved in dressing swine carcasses were then evaluated in the second experiment. A set of 40 randomly selected carcasses leaving singeer, polisher, shaver, and washer were sampled. Carcasses were heavily contaminated during the final polishing procedure, and the APC increased threefold compared with prepolishing levels. Washing reduced the bacterial numbers by 69%. To reduce the microbial load on swine carcasses, final polishing and manual shaving steps were not used during the dressing operation on a set of 90 carcasses. APCs on singed carcasses were reduced from 1.34 to -0.15 log10 CFU/cm2 when the final polisher and manual shavers were not used. However, carcasses were subsequently recontaminated with bacteria after evisceration, and the APCs were similar (P > 0.05) regardless of whether the final polishing and manual shaving steps were used, averaging 1.30 and 1.46 log10 CFU/cm2. These results indicated that individual operations can be identified as critical control points, appropriate limits can be set and monitored in a hazard analysis and critical control point system, and steps where further changes to reduce bacterial levels may be needed for swine slaughtering plants.