Cell Culture and Protein Detection

Cell Culture and Protein Detection Overview: This practical is split into three main parts, each part allows us to develop certain techniques. The first part of the practical is focused on the techniques needed to successfully carry out cell cultures. The second part is using an analytical technique known as ELISA this is used to measure the amount of proteins secreted by the cells that were cultured in part one. Lastly, part three focuses on western blotting, this is a technique used to measure proteins too but it differs from ELISA in that it measures proteins that are present inside the cell. The fundamental objective of this practical is to look at how Bacterial lipopolysaccharide (LPS) alters the growth and the expression of the smooth muscle cell ÃŽ ±-actin (SMA) in vascular smooth muscle cells (vSMC) while also detecting and quantifying cell signalling molecules (JNK’s) and cytokine secretion (TNF-ÃŽ ±) in vascular cells. Section 1 – Subculture Objective: The aim of this part of the practical is to subculture the bovine aortic vascular smooth muscle cells and to count the cells and check for viability by counting the cells in suspension on a haemocytometer using a microscope. The cells are also treated with a bacterial antigen (LPS) to look at its effect on viability, differentiation state and the activation of intracellular signalling and secretion of proteins. Immunocytochemistry is also carried out in this part of the practical. Method: Trypsin is used to remove the cells from the bottom of the flask. When the cells have dissociated, medium which contains a trypsin inhibitor is added. The medium stops excessive trypsin action from damaging the cells. The cell suspension is first diluted using trypthan blue to allow viable cells which remain white to be distinguished from non- viable cells which are blue. This method allows us to get a total cell count of cells/mL and the percentage of viable cells. Our total cell count was found to be 7.45105 cells/mL The non-viable cells were found to be 1.5104 cells/mL This means that the culture contains more 98% viable cells which indicates that it is a healthy culture. 3 different 6 well plates were then set up using different densities of seed cells. 3105cells/ml seeding density was used in one plate. This density was used as a lot of protein needs to be present for the assessment of JNK and ÃŽ ±-actin protein cytokine production. 1105 cells/ml seeding density was used in another 6 well plate. These plates are used to assess the effect of LPS on growth and viability of the cells. This density is used to give a clear picture of the cells to make counting easier. 5103 cells/ml seeding density was used in the last plate. This plate is used for ÃŽ ±-actin expression by immunocytochemistry. The low density will give us a clearer picture. The three different sets of plates are all treated with varying concentrations of LPS. Each plate has two wells which are used as controls, containing no LPS, two wells containing 1Â µg of LPS and two wells containing 10Â µg of LPS. Immunocytochemistry is carried out on the plate with a seeding density of 5103cells/ml. This technique is used to determine if a particular protein or antigen is present. An unlabelled primary antibody is used to bind to the antigen desired antigen. The presence of contractile protein ÃŽ ±-actin can determine the differentiation state of vSMC. Immunocytochemistry is used as a qualitative method of determining the presence of a protein, it is not quantitative. Results: Calculations for different seeding densities: Initial concentration = 7.45105 cells/mL Formula = Plate 1, seeding density 1.5105 cells/ml Plate 2, seeding density 0.5105 cells/ml Plate 2, seeding density 2.5103 cells/ml Table 1: Cell growth for each group Immunochemistry results: Figure 1: Cell growth in the control (0Â µg/ml LPS) Figure 1: Cell growth in the control (1Â µg/ml LPS) Figure 3: Cell growth with 10Â µg/ml of LPS Discussion: The first part of this section was to carry out a cell count and determine the viability of the suspension. Our sample had 7.45105 cells/ml and it contained more than 98% viable cells. This meant it was a healthy cell suspension and it was suitable to run tests on for the practical. A seeding density of 1105 cells/ml seeding density was used to assess the effect of LPS on growth and viability of the cells. This density was used to give a clear picture of the cells to make counting easier. The results of our experiment correlated with the majority of the class. However there were some unexpected results in some of the groups but this could be down to plates being labelled incorrectly or mistakes while counting when using the haemocytometer. Immunocytochemistry is used to allow us to analyse the results visually. We found that as the amount of LPS increased the number of cells decreased, this is illustrated in figures 1, 2 and 3. LPS is an endotoxin and it inhibits the growth of ÃŽ ±-a ctin. LPS also damages the structure of ÃŽ ±-actin, high levels of LPS stop the correct formation of the filaments and so affects the function of the cell which is to facilitate cell contraction and migration. This result was expected as LPS is a major mediator to septic shock and is known to directly affect vascular smooth muscle cells. Question: What could you do to improve this experiment? To improve this experiment I would use a wider range of concentrations for LPS. This would give a better understanding of its affects. Repeating the experiment several times and getting an average of your results would also help. Section 2 – ELISA Objective: The aim of this section of the practical is to use Enzyme-Linked Immunosorbent Assay (ELISA) to detect production of the cytokine TNFÃŽ ± from the cells activated with LPS. Detection is based on a colour change. Firstly a standard curve must be generated so that absorbance values can be converted into concentrations of TNF-ÃŽ ±. Once the curve is completed we can determine the unknown concentrations of TNF-ÃŽ ± in our samples. Method: Firstly the antibody is immobilised onto the surface of the plate. The plate is then washed to remove any excess antibody, antigen is then added and it is allowed to bind to the antibody. A secondary antibody is then added, this antibody is labelled with an enzyme. The enzymes substrate is then added, this causes a colour change. The amount of coloured product formed is determined spectrophotometrically. The amount of coloured product is proportional to the amount of enzyme present and also to the concentration of the antigen. Results: Table. 2 Absorbance values of samples at 450nm. Figure 4. Plot of standard curve of absorbance versus concentration. Discussion: The aim of this experiment was to quantify the amount of TNF-ÃŽ ± present in our samples. To do this a set of known standards were used and their absorbance values read. This data produced a straight line with an R2 value of 0.99 indicating that a straight line was an excellent fit for absorbance versus concentration, and so the equation of the line could be used to determine unknown concentrations of TNF-ÃŽ ± based on their absorbance values. Our results showed that TNF-ÃŽ ± was present in its highest concentration of 25.15pg/ml in the sample with the highest concentration of LPS and it was found in its lowest concentration of 9.9 pg/ml in the sample containing no LPS. This result was expected as cytokines such as TNF-ÃŽ ± are produced in large quantities to respond to endotoxins such as LPS. Question: What could you do to gain more information from this experiment? To gain more information from this experiment you could test for other cytokines which are also activated by LPS, correlating these results would make your data more meaningful. Section 3 – Western Blotting Objective: The aim of this section of the practical is to prepare cell lysates from the vascular smooth muscle cells which were activated by LPS previously. SDS PAGE and western blotting will then be used to detect the activation of the intracellular protein JNK. Western analysis quantifies the amount of protein present in the cell. To do this cell lysis must be carried out. In this practical we used a method which generates whole cell lysates. To do this lysis buffer is added to the cells followed by sonication. Method: Cell lysis is carried out first. The next step is SDS polyacrylamide gel electrophoresis. Western blotting involves transferring the protein bands from an acrylamide gel to a more stable and immobilising medium such as nitrocellulose paper so that analytical procedures such as detection with antibodies can be carried out. We carried out western blotting using iBlot dry blotting system. After blotting probing is carried out to determine the presence of phosphorylated JNK protein. Results: Figure 5. Ponceau S Staining Figure 6. ÃŽ ±-actin Figure 7. pJNK Discussion: To see if our transfer was a success before probing, the blot was stained with Ponceau S stain. The proteins can be seen as red bands with this stain. The result of this stain can be seen in figure 5. red bands are present which indicates our transfer was a success and that there are proteins present. The western blot analysis showed that ÃŽ ±-actin was present in all the samples as a strong band around 42kDa was observed which is expected for ÃŽ ±-actin. The results for pJNK did not work out as it was washed incorrectly, because of this no bands were observed, however bands would be expected in the samples containing LPS. Why did you run the sample on the gel before blotting? The sample was run on gel first as proteins are separated by molecular weight. This allows us to distinguish ÃŽ ±-actin from other proteins. It is then moved to the nitrocellulose paper so that analytical procedures such as detection with antibodies can be carried out Why measure the phosphorylated form of JNK? The phosphorylated form of JNK is a signal a cell sends out when it is stressed. Therefore pJNK should be present in the samples with LPS. If it is present then it confirms the fact that the cell is stressed as a result of the presence of the endotoxin.

Depression and Finding Help Essay -- Depression Psychiatry Psychology

Depression and Finding Help Depression is defined as an illness; the feelings of depression persist and interfere with a child or adolescent’s ability to function. Depression can be a very difficult and painful experience that affects not only the individual suffering from it, but also the people around them. There comes a point in some peoples’ lives where social isolation, low energy, sadness, low self-esteem, and the feeling of hopelessness, cannot be taken anymore. The feelings are so strong and persistent, that the victim becomes severely unhappy, which can then result in depression. Clinical depression has many related symptoms trouble sleeping, eating disorders, withdrawal and inactivity, self-punishment, and loss of pleasure. People that are depressed do not like to do things they may usually like to. However, there are many differences between feeling depressed and actually suffering from depression, the disease. Any person feels depressed at some time or another in their life. They feel worthless, tired and tend to want to be alone without human interaction, this is normal. Depression brings on poor concentration or inability to think and make decisions (Kist 26). Surveys that have been taken that show approximately 20 in 100 people suffer from depression at any one time. About one if four Americans will suffer from a depression over the course of their lifetime. Depression strikes men and women of all ages, in all races, but most studies indicate that women are more often afflicted. Depression comes in many different types: major, manic and dysthymia are a few. A chronic, physical illness, drug habit, death of a loved one; or a problem in a marriage cause major depression. Even though most people with major depression will recover, half will suffer another episode (Hales 38). People with severe cases of major depression can’t work, study, or interact and eventually can’t feed, clothe or clean themselves (Hales 38).Manic depression is a type of depres sion that goes from a person being extremely happy and then becoming severely depressed (Kist 107). Being in a depressed state can be life threatening. People suffering from manic depression show many symptoms. A few major ones are hyperactivity; talking fast, inability, fear of dying, and jumping from one topic to another during a conversation (Kist 39). Another type is Psychoanalysis. Psychoanaly... ...on to a better, more productive lifestyle. Overall, there are many causes of this unbearable disease, and often many times a combination of genetic, psychological, and environmental factors are involved in the onset of a depressive disorder. However, the big risk factor in major depression is suicide. Within five years of suffering from major depression, an estimated 25% of sufferers try to kill themselves. Therefore, it is important to take action when confronted with the symptoms of this disease. Depression reaches many people and that is why I chose this as my topic. Depression is maybe the most painful illness, but it can be overcome. Hopefully everyone with depression seeks help either through medication or therapy. Bibliography Blackman, Maurice. You Asked about †¦ Adolescent Depression. Available at http://www.mental.health.com/mag1/pgs51-dp01.html Hales, Dianne. Depression. Chelsea House Publishers, New York, Philadelphia, 1989. Kist, Jay. "Dealing with Depression† Current Health 2 Jan.1997 25-28. Lytle, Victoria. The dark clouds of Depression. NEA Today Dec.1994. Spike Milligan and Anthony Clare. Depression and how to Survive it. Arrow 1994.

Succubus Blues CHAPTER 25

Sometimes you wake up from a dream. Sometimes you wake up in a dream. And sometimes, every once in a while, you wake up in someone else's dream. â€Å"If he wanted to carry me off and make me his love slave, I'd do it, so long as I got advance copies of his books.† My first words spoken to Seth as I passionately discussed his work. Seth's initial impression of me. Head held high, hair tossed over my shoulder. A flippant remark always at the ready. Grace under fire. A cool social confidence introverted Seth could never muster but envied. How can she do that? Never miss a beat? Later, my rambling explanation of the five-page rule, a goofy habit he found infinitely endearing. Someone else who appreciated literature, viewing it like fine wine. Smart and deep. And beautiful. Yes, beautiful. I saw myself now as Seth had seen me that night: the short skirt, the racy purple top, brilliant as a bird's plumage. Like some exotic creature, hopelessly out of place in the bookstore's dreary landscape. All of this was in Seth, the past of his growing feelings for me mingling with the present, and I drank everything up. Not just beautiful. Sexy. Sensual. A goddess made flesh whose every move hinted at passion to come. The dress strap slipping off my shoulder. Faint beads of perspiration on my cleavage. Standing in his kitchen, clad only in that ridiculous Black Sabbath shirt. No underwear on under that. Wonder what it'd be like to wake up with her next to me, messy and untamed. It all spilled into me. More and more. He would watch me at the bookstore. Loved watching me interact with customers. Loved that I seemed to know something about everything. The witty dialogue he pondered for his characters coming to my lips without hesitation. Amazing. Never met anyone who talks like that in real life. My bartering with the used book store owner. A charisma that drew in shy, quiet Seth, made me glow in his eyes. Made him feel more confident. Still his feelings rushed through me. I had never felt anything like it. Certainly I had felt attraction and fondness in my victims, but never such love, not directed at me. Seth thought I was sexy, yes. Desired me. But that raw lust juxtaposed with something softer too. Something sweeter. Kayla sitting on my lap, small blond head against my chest as I braided her hair. A brief shifting of the image as he momentarily considered his own daughter on my lap. Fierce and witty on one hand, gentle and vulnerable on the other. My inebriated state at his condo. A swell of protectiveness as he led me to bed, watching me hours after I'd gone to sleep. He thought no less of me for the weakness, for my lapse of control and judgment. It was a letting down of my walls for him, a sign of imperfection that made him love me more. Further and further I drank, my desperate and weakened state unable to stop. â€Å"Why doesn't she date?† Seth asked Cody. Cody? Yes, there he was, in the back of Seth's mind. A memory. Cody secretly giving Seth swing lessons, neither of them telling me, instead making up vague excuses for why they always had to be â€Å"somewhere.† Seth, trying so hard to make his feet obey so he could dance with me and be closer to me. â€Å"She's afraid,† the vampire replied. â€Å"She thinks love causes pain.† Love causes pain. Yes, Seth loved me. Not the crush I'd imagined. Not a superficial attraction I thought I'd dissuaded. It was more, so much more. I embodied everything in a woman he could ever imagine: humor, beauty, intelligence, kindness, strength, charisma, sexuality, compassion†¦ His soul seemed to have recognized mine, drawn uncontrollably toward me. He loved me with a depth of feeling I could not even begin to tap into, though believe me, I tried. I wanted it. I wanted to feel it all, to suck up that burning within him. To consume it. Set myself on fire with it. Georgina! Somewhere far away, someone called to me, but I was too into Seth. Too into absorbing that strength within him, that strength fused with his feelings for me. Feelings brought on, amplified even, by kissing. Lips soft and eager. Hungry. Demanding. Georgina! I wanted to become one with Seth. I needed to. I needed him to fill me up†¦ physically, mentally, spiritually. There was something there†¦ something concealed inside him I couldn't quite reach, hovering in the background. A tantalizing piece of knowledge I should have long since recognized. You are my life. I needed to get in farther, reach out for more. Find out what was hiding from me. That kiss was my lifeline, my connection with something bigger than myself, something I had been aching for all my life but never known. I couldn't stop. Couldn't stop kissing Seth. Couldn't stop. Couldn't – â€Å"Georgina! Let go!† Rough hands tore me away from Seth, like flesh ripping from my own body. I cried out in agony at the broken connection, fighting the hands that pulled me and held me. I clawed at my captor, needing to find out the secret lurking beyond that kiss, yearning for the completeness of that union with Seth – Seth. My hands dropped, and I blinked, bringing the world back into focus. Reality. I was no longer inside Seth's head; I was still in my apartment. A feeling of solidness settled in me, and I didn't have to look down to know my body had stopped its shifting, my form snapping back to a short, slim woman with honeyed brown hair. The girl I had been long ago was buried within me once more, never to come out if I could help it. Seth's life force now filled me to overflowing. â€Å"Georgina,† murmured Hugh behind me, letting his hands ease up on my arms. â€Å"Christ, you scared me.† Looking across the room, I saw Carter, bedraggled as usual, leaning over Seth's body. â€Å"Oh God – † I sprang up and moved to them, kneeling beside the angel. Seth lay on the floor, skin pale and clammy. â€Å"Oh God. Oh God. Oh God. Is he†¦ ?† â€Å"He's alive,† Carter told me. â€Å"Barely.† Stroking Seth's cheek, feeling the fine golden-red haze of his near-beard, I felt tears brimming in my eyes. His breathing came shallow and jagged. â€Å"I didn't mean to. I didn't mean to take so much†¦Ã¢â‚¬  â€Å"You did what you had to do. You were in bad shape, could have died.† â€Å"And now Seth might†¦Ã¢â‚¬  Carter shook his head. â€Å"No. He won't. He'll need recovery time, but he'll pull through.† I drew my hand back, half-afraid my touch might harm Seth more. Glancing around, I became aware of the disheveled state of my apartment. It looked worse than Jerome's. Smashed china and glass. Broken tables. Overturned chairs and couch. The unstable bookshelf in pieces at last. From the kitchen, Aubrey hunkered down under the kitchen table, wondering what was going on. I wondered myself. The nephilim were nowhere in sight. What had happened? Had I really missed it all? The epic, divine battle of the century, and I had missed it for a kiss? Admittedly, a really good kiss, but still†¦ â€Å"Where is†¦ everyone else?† â€Å"Jerome's off doing, uh, damage control with your neighbors.† â€Å"That doesn't sound good.† â€Å"Standard practice. Supernatural battles aren't exactly quiet, you know. He'll do a little mind erasing, make sure no authorities get notified.† I swallowed, afraid to ask my next question. â€Å"What about†¦ what about the nephilim ?† Carter studied me, gray eyes holding me long and hard. â€Å"I know, I know,† I said at last, looking down, unable to return that gaze. â€Å"There's no ten years and parole, right? You destroyed them.† â€Å"We destroyed†¦ one of them.† I looked up sharply. â€Å"What? What about the other one?† â€Å"He got away.† He.My looming tears slipped out now; I could not control them. For you, I'll walk away. â€Å"How?† Carter laid a hand on Seth's forehead as though taking vital stats and then turned back to me. â€Å"It all happened really fast. He masked and went invisible in the confusion, while we were taking on the other one. And honestly†¦Ã¢â‚¬  The angel looked at my closed front door, then at Hugh and me. â€Å"What?† I whispered. â€Å"I'm not†¦ I'm not entirely convinced Jerome didn't let him get away. He wasn't expecting two. I wasn't either, though I should have, in retrospect. After killing the first one†¦Ã¢â‚¬  Carter shrugged. â€Å"I don't know. Hard to say what happened. â€Å" â€Å"Then he'll be back,† I realized, fear and relief blending weirdly in me over the thought of Roman's escape. â€Å"He'll be back†¦ and he won't be happy with me.† â€Å"I don't think that'll be a problem,† the angel observed. Gently, he lifted Seth up and walked to my overturned couch. A moment later, it flipped over untouched, righting itself. Carter laid Seth on it and continued speaking. â€Å"He took a real beating – the other nephilim. A really bad beating. I can't believe he had the power left to hide himself from us; I still keep expecting to feel him again any minute. If he's smart, he's running as fast and far from us as he can right now, getting out of our range – out of any immortal's range – so he can drop his shields and rest.† â€Å"Then what?† asked Hugh. â€Å"He's in bad shape. It'll take him a long time to recover. When he does, he knows he doesn't have the backup to return here again.† â€Å"He could still take on me,† I noted, shivering as I remembered Roman's wrath toward me at the end. It was hard to believe we'd been wrapped in each other's arms, caught in the throes of passion, less than twenty-four hours ago. â€Å"He could take you on,† agreed Carter. â€Å"But he can't take me on. Or Jerome. He certainly can't take both of us on. That was what decided it, in the end. They didn't expect that. Us teamed together. It'll give him pause to just come bursting back here, even if you alone pose no threat.† I didn't find that reassuring in the least. I thought of Roman, passionate and rebellious, always eager to make a point against the system. That personality type lent itself well to revenge. I had tricked him, made love to him, and then betrayed him, resulting in the annihilation of his plans – and his sister. Thank God for my sister. She's the only one I have, the only mainstay in my life. He might pause, as Carter had suggested, but not for long. Of that, I was certain. â€Å"He'll be back,† I whispered, more to myself. â€Å"Someday he'll be back.† Carter gave me a steady look. â€Å"Then we will deal with him then.† My front door opened, and Jerome entered. He looked neat and prim, hardly like he'd just been in an apocalyptic battle with his own offspring. â€Å"Housekeeping all done?† asked Carter. â€Å"Yes.† The demon's eyes darted over to Seth. â€Å"He's alive?† â€Å"Yes.† Angel and demon locked eyes then, and a tense moment of palpable silence hung between them. â€Å"How fortuitously unexpected,† Jerome murmured at last. â€Å"I could have sworn he was dead. Well. Miracles happen every day. I suppose we'll have to wipe him now.† I stood up. â€Å"What are you talking about?† â€Å"Nice to have you back with us again, Georgie. You look lovely, by the way.† I glared at him, angry at his joke, knowing it was Seth's energy giving me the succubus glamour now. â€Å"What do you mean you have to ‘wipe' him?† â€Å"What do you think? We can't let him walk away after everything he's seen. I'll diminish a little of his affection for you while I'm at it; he's a liability to you.† â€Å"What? No. You can't do that.† Jerome sighed, putting on the look of one who suffered long and hard. â€Å"Georgina, do you have any idea what he was just exposed to? He has to be wiped. We can't let him know about us.† â€Å"How much of me will you take from him?† Pieces of Seth's memories – my memories, now – glittered in my head like jewels. â€Å"Enough so that he forgets he has any more than a passing knowledge of you. You've been even more negligent with your job than usual these last few weeks.† I hardly thought that was Seth's fault; Roman had helped too. â€Å"Both of you will function much better if he finds some mortal woman to obsess on instead.† Don't you want to stand out in some way? Carter's taunting question from what seemed like an eternity ago whispered in my head. â€Å"You don't have to do this. You don't have to take me out with the rest.† â€Å"If I'm already in there, I might as well clear you too. There's no way he can just go on as usual after being exposed to denizens of the divine realms. Even you have to agree with that.† â€Å"Some mortals know about us,† I argued. â€Å"Like Erik. Erik knows, and he keeps it to himself.† In fact, I realized suddenly, Erik had kept Helena's secret to himself as well. He had figured it out after working with her over the years but had never revealed the full truth, only doling out small clues for me. â€Å"Erik is a special case. He has a gift. An ordinary mortal like this one couldn't handle it.† Jerome walked over to my couch, looking at Seth dispassionately. â€Å"It's better this way.† â€Å"No. Please,† I cried, running over to Jerome and pulling his sleeve. â€Å"Please don't.† The archdemon turned to me, dark eyes cold and shocked that I would dare grab hold of him like that. I knew then, cringing under that gaze, that something in our fond, indulgent relationship had changed forever – something small, but important nonetheless. I didn't know what had done it. Maybe it had been Seth. Maybe it had been Roman. Maybe it had been something else altogether. All I knew was that it had happened. â€Å"Please,† I begged, ignoring how desperate I must sound. â€Å"Please don't. Don't take me from him†¦ out of his head like that. I'll do anything you want. Anything.† I brushed a hand over my eyes, attempting to look calm and in control, knowing I was failing. One eyebrow shifted ever so slightly on Jerome's face, the only hint that I had piqued his interest. The term â€Å"deal with the devil† had not arisen lightly; few demons could resist a bargain. â€Å"What could you possibly offer me? The sex thing only worked on my son, so don't even think about trying it now.† â€Å"Yes,† I agreed, voice growing stronger as I plunged forward. â€Å"It worked on him. It works on all sorts of men. I'm good, Jerome. Better than you know. Why do you think I'm the only succubus in this city? It's because I'm one of the best. Before I hit this funk†¦ this, I don't know, whatever mood I've been in for a while now, I could have any man I wanted. And not just simply for their strength and life force. I could manipulate them. Make them do anything I asked, talk them into acts of sin they never would have dreamed of before meeting me. And they would do it. They'd do it, and they'd like doing it.† â€Å"Go on.† I took a deep breath. â€Å"You're tired of the ‘all lowlifes, all the time,' right? Me being negligent? Well, I can change that. I can raise your stock higher than you've ever dreamed. I've done it before. All you have to do is let Seth go. Let him keep his memories intact. All of them.† Jerome studied me a moment, mind working. â€Å"All the ‘stock' in the world won't do me any good if he runs around blathering about what he's seen.† â€Å"Then we'll see if he can handle it first. When he recovers and wakes up, we'll talk to him. If he doesn't look like he'll be able to cope with it all†¦ well, then you can erase his memories.† â€Å"Who will make the call if he can cope or not?† I hesitated, not wanting that decision in the demon's hands. â€Å"Carter will. Carter can tell if someone's telling the truth.† I looked at the angel. â€Å"You'll know if it's okay, right? Okay for him to know†¦ about us?† Carter gave me an odd look, one I could not interpret. â€Å"Yes,† he finally admitted. â€Å"What about your end?† asked Jerome. â€Å"Will you hold it up – even if Carter decides he's unsafe?† That was harsh. I had a feeling Jerome wouldn't negotiate on this one, but I was willing to risk it, so confident did I feel about Seth's capacity to process immortal activity. I opened my mouth, about to agree, when I caught Hugh shaking his head at me out of the corner of my eye. Frowning, he tapped his watch, mouthing something I couldn't understand at first. Then, it clicked. Time. I had listened to the imp talk about his job enough to know the rules of negotiation: never make an open-ended deal with a demon. â€Å"If Seth keeps his memories, I'll walk the succubus straight and narrow for a century. If they have to be erased anyway, then I'll still do it for†¦ a third of that.† â€Å"Half,† countered Jerome. â€Å"We aren't mortal. Even a century is nothing on the face of eternity.† â€Å"Half,† I agreed dully, â€Å"but no more than survival dictates. I'm not going to do this every day, if that's what you're thinking. I'll only get fixes as I need them, but they'll be strong ones. Very strong – loaded with sin. With men of good caliber, that'll be†¦ oh, every four to six weeks.† â€Å"I want better than that. Extra credit. Every couple of weeks, whether you need it or not.† I closed my eyes, unable to fight anymore. â€Å"Every couple of weeks.† â€Å"Very well,† said Jerome, a warning note in his voice. â€Å"But you will be held to this agreement unless I choose to terminate it for some reason. Not you. There will be no wiggling out for you.† â€Å"I know. I know, and I accept.† â€Å"Shake then.† He extended his hand to me. Not hesitating, I took it, and power crackled briefly around us. The demon smiled thinly. â€Å"We have a deal.†

Dinah and Male-Dominated Biblical Narrative

One of the aptest historical criticisms of The Holy Bible is the way it fails to chronicle womens lives, abilities and viewpoints with the same effort it puts into mens lives. The story of Dinah in Genesis 34 is one of the best examples of this male-dominated narrative. A Young Woman at the Mercy of Men Dinahs story actually starts in Genesis 30:21, which tells of her birth to Jacob and his first wife, Leah. Dinah reappears in Genesis 34, a chapter that early versions of the Bible titled the rape of Dinah. Ironically, Dinah never speaks for herself in this significant episode of her life. In brief, Jacob and his family are encamped in Canaan near the city of Shechem. By now having reached puberty, teen-aged Dinah understandably wants to see something of the world. While visiting the city, she is defiled or outraged by the prince of the land, also called Shechem, who is the son of Hamor the Hivite. Although scripture says Prince Shechem is eager to marry Dinah, her brothers Simeon and Levi are enraged at the way their sister has been treated. They convince their father, Jacob, to exact a high bride price, or dowry. They tell Hamor and Shechem that it is against their religion to allow their women to marry men who are not circumcised, i.e., converts to the religion of Abraham. Because Shechem is in love with Dinah, he, his father, and eventually all the men of the city agree to this extreme measure. However, circumcision turns out to be a trap devised by Simeon and Levi to incapacitate the Shechemites. Genesis 34 says they, and possibly more of Dinahs brothers, attack the city, kill all the men, rescue their sister and despoil the town. Jacob is horrified and frightened, fearing that other Canaanites sympathetic with the people of Shechem will rise against his tribe in retaliation. How Dinah feels at the murder of her betrothed, who by this time may even have been her husband, is never mentioned. Rabbinical Interpretations Vary on Dinahs Story Later sources blame Dinah for this episode, citing her curiosity about life in the city as a sin since it exposed her to risk of rape. Shes also condemned in other rabbinical interpretations of scripture known as Midrash because she didnt want to leave her prince, Shechem. This earns Dinah the nickname of the Canaanite woman. A text of Jewish myth and mysticism, The Testament of the Patriarchs, justifies the anger of Dinahs brothers by saying that an angel instructed Levi to take revenge on Shechem for the rape of Dinah. A more critical view of Dinahs story holds the tale may be not historical at all. Instead, some Jewish scholars think Dinahs story is an allegory that symbolizes the way Israelite men conducted feuds against neighboring tribes or clans that raped or abducted their women. This reflection of ancient customs makes the story valuable, according to Jewish historians. A Feminist View of Dinahs Story In 1997, novelist Anita Diamant re-imagined Dinahs story in her book, The Red Tent, a New York Times best-seller. In this novel, Dinah is the first-person narrator, and her encounter with Shechem is not rape but consensual sex in anticipation of marriage. Dinah willingly marries the Canaanite prince and is horrified and grieved by her brothers vengeful actions. She flees to Egypt to bear Shechems son and is reunited with her brother Joseph, now Egypts prime minister. The Red Tent became a worldwide phenomenon embraced by women who longed for a more positive view of women in the Bible. Although entirely fiction, Diamant said she wrote the novel with attention to the history of the era, around 1600 B.C., particularly in terms of what could be discerned about the lives of ancient women. The red tent of the title refers to a practice common to tribes of the ancient Near East, in which menstruating women or women giving birth lived in such a tent along with their co-wives, sisters, daughters and mothers. In a question-and-answer on her website, Diamant cites work by Rabbi Arthur Waskow, who links the biblical law that keeps a mother separate from the tribe for 60 days upon the birth of a daughter as a sign that it is a sacred act for a woman to bear to another potential birth-giver. A subsequent work of non-fiction, Inside the Red Tent by Baptist scholar Sandra Hack Polaski, examines Diamants novel in light of both biblical story and ancient history, particularly the difficulties of finding historical documentation for womens lives. Diamants novel and Polaskis non-fiction work are completely extra-biblical, and yet their readers believe that they give voice to a female character whom the Bible never allows to speak for herself. Sources Giving Voice to Dinah Sermon given December 12, 2003, by Rabbi Allison Bergman Vann The Jewish Study Bible, featuring the Jewish Publication Societys TANAKH translation (Oxford University Press, 2004). Dinah by Eduard KÃ ¶nig, Emil G. Hirsch, Louis Ginzberg, Caspar Levias, Jewish Encyclopedia. Ten Questions on the Occasion of the Tenth Anniversary of The Red Tent by Anita Diamant (St. Martins Press, 1997). Inside the Red Tent (Popular Insights) by Sandra Hack Polaski (Chalice Press, 2006)