If a crystalloid solution is used N as the bypass prime purchase genuine malegra fxt plus line impotence statistics, the consequent dilution of the patient’s H+ blood will result in a signifcant reduction in the nonbicar- 7 cheap malegra fxt plus 160mg free shipping diabetes and erectile dysfunction causes. Erythrocyte dilution to a hematocrit of 20% purchase malegra fxt plus with amex erectile dysfunction age 33, associated with the same degree of plasma dilution, results in a decrease in buffer strength of approximately 61 7. If the hematocrit is reduced to between 24 and 28% dur- ing bypass, this will result in a 20% reduction in nonbi- carbonate buffering. Hypothermia Phosphate As noted earlier in this chapter, the pH of water increases 6. Another way of expressing this fact is that the pH of neutrality (pN) rises with hypothermia. The pH which is read from the machine can be temperature corrected to a 0 10 20 30 40 body temperature of X°C by the formula: Temperature (°C) pHxºC = –pH37°C + (37 – x)(0. Because the imidazole moiety It is interesting to note that the fact that the slope of the of the amino acid histidine in proteins performs the bulk of buffer- change in pH with temperature of whole blood is similar to ing in blood, the slope of the dissociation constant for blood is simi- that for neutral water refects the dominant role of imidazole lar to the slope of the dissociation constant for water. The importance of acid-base man- stant (pK) for imidazole is very similar to the dissociation agement for cardiac and cerebral preservation during open heart constant for water (Fig. The pH of neutrality of blood retains during Cardiopulmonary Bypass its usual alkalinity relative to intracellular pH so that there Ectothermic (“cold-blooded”) animals and hibernating mam- is a constant hydrogen ion concentration gradient between mals have provided an opportunity for study of the alternative the intracellular and extracellular environments both at nor- methods whereby different species adjust their physiology mothermia and hypothermia. Interestingly dif- temperature allows these animals to maintain a constant ratio ferent strategies have evolved for species that must remain of hydrogen to hydrogen ions across a wide range of tempera- active while hypothermic versus those that hibernate. They are faced with the problem “alpha”) of dissociated to nondissociated imidazole groups of needing to be able to mobilize energy stores effciently remains constant with this strategy. They achieve optimal energy mobilization by maxi- Hibernators and the pH Stat Strategy mizing enzyme effciency. As their temperature falls, these Hibernating mammals (heterotherms) follow a diametri- animals allow both intracellular pH and extracellular pH cally opposite strategy to the maintenance of acid-base dur- to increase parallel with the rise in the pH of neutrality of ing hypothermic hibernation relative to ectotherms. If this blood gas sample (“luxuriant”) cerebral blood fow with the potential for an is read at 37°C, the pH will be 7. This degree of In contrast, the use of alpha stat pH management pre- acidosis renders the intracellular environment too acidic for served cerebral autoregulation thereby allowing maintenance optimal enzymatic activity, thereby depressing metabolism of coupling between fow and metabolism down to low tem- and preserving intracellular substrates. Interestingly, it appears extend the lower limit of cerebral autoregulation down to a that the regulation of intracellular pH in hibernators does not mean arterial pressure of 30 mm (cerebral perfusion pressure necessarily conform to the pattern of pH stat in all organs. Although one of the four trials demonstrated no differ- an alkaline direction during hypothermia following a pattern ence in adults undergoing moderately hypothermic relatively similar to the alpha stat system. Traditional management of acid-base balance during cardio- In 1985, we changed our pH strategy for hypothermic cardio- pulmonary bypass in the 1960s and 1970s was to follow the pulmonary bypass at Children’s Hospital Boston to the more pH stat model. Before 1985, we had used the more mathematically temperature corrected using a nomogram acidotic pH stat strategy. Like many other units at that time, to patient temperature and carbon dioxide was added to the our rationale for changing our pH management was based oxygen passing through the oxygenator to ensure that the solely on comparative physiological studies in cold-blooded temperature-corrected arterial pH remained at 7. First, Retrospective Clinical Study of pH Strategy In order to it was suggested that the leftward shift of the oxyhemoglo- determine the impact of the change in pH strategy on our bin dissociation curve that occurs during hypothermia (with infant patient population undergoing circulatory arrest, we the consequent increased affnity of hemoglobin for oxygen), undertook a retrospective developmental study with a cohort would be reversed by the addition of carbon dioxide which of patients who had undergone surgery for transposition shifts the curve to the right and increases tissue oxygen avail- in a timeframe that straddled our change in pH strategy. Second, cerebral vasodilation due to carbon dioxide Sixteen patients who had undergone Senning procedures was thought to be benefcial in increasing cerebral blood fow between 1983 and 1988 underwent cognitive developmen- selectively during the stressful period of cardiopulmonary tal assessment. The mean began to appear describing the alpha stat strategy of pH man- scores were not associated with any patient-related vari- agement utilized by ectotherms as described above. The duration of circulatory arrest was 43 minutes, fact that the alpha stat strategy maintained optimal enzyme ranging from 35 to 60 minutes. Duration of circulatory activity was an appealing rationale to change from the pH arrest was not associated with cognitive outcome. For example, there were many changes in demonstrated that the use of pH stat during cardiopulmonary perfusion technique during this time, such as a change from 194 Comprehensive Surgical Management of Congenital Heart Disease, Second Edition pH In vivo 8. It can be seen that rewarming of a blood gas specimen by the blood gas analyzer results in a pC02 reading of 156 mm. The importance of acid-base management for cardiac and cerebral preservation during open heart operations. Between 1986 and 1990, following the introduction Decreased cerebral blood fow during cooling may have of the alpha stat strategy in 1985, 19 cases of choreoathetosis resulted in inadequate, nonhomogeneous brain cooling occurred. Most of the children who developed choreoathetosis had complex Laboratory Studies of pH Strategy In order to better forms of pulmonary atresia with multiple collaterals. They understand the basic mechanisms behind the fndings of our also underwent deep hypothermic circulatory arrest. We clinical studies, we undertook two separate studies using Conduct of Cardiopulmonary Bypass 195 more acidotic during early reperfusion than it had become 140 during the circulatory arrest period itself, while it showed immediate recovery with reperfusion with pH stat. A particularly inter- esting fnding of this study was the redistribution of cerebral 100 blood fow during cooling, which was different between the two groups (Fig. Animals undergoing alpha stat had 80 a decrease in the proportion of blood perfusing the basal ganglia in addition to having (as expected) less total cere- bral blood fow than the pH stat animals. However, even with the shift in distribution, underwent deep hypothermic circulatory arrest between 1983 the absolute regional blood fow to the cerebral hemispheres and 1988, there was a strong positive correlation between arte- was not less with pH stat than alpha stat. J Thorac Cardiovasc Surg latory arrest which could contribute to an improved cerebral 1993;106:362–8. In the initial acute study, we found that epidemic of choreoathetosis in the late 1980s after our intracellular pH determined by magnetic resonance spec- change to alpha stat, our retrospective clinical trial sug- troscopy showed an alkaline shift during core cooling in gesting worse developmental outcome with alpha stat and both groups, but became more alkaline with alpha stat than the consistent fndings from our laboratory studies sug- pH stat at the end of cooling (p = 0. In 1992, we began a randomized, prospective the initial 30 minutes of reperfusion was faster with pH stat. Early mortality occurred in four tion to supplying oxygen and other substrates, cerebral infants (2%), all in the alpha stat group. Thus, even mortal- blood fow is also the principal means by which the ity itself was lower with pH stat at close to a 0. In fact, the total volume of occurred in fve of 57 patients (9%) assigned to alpha stat blood needed to cool the brain to the desired deep hypo- and one of 59 patients (2%) assigned to pH stat (p = 0. Thus, ity returned sooner among infants randomized to pH stat (p the pH stat strategy should be preferred not only for the = 0. This is important this result was strongly infuenced by one outlier who was not in the early cooling phase when the brain is tested for microdeletion of chromosome 22. Studies from Other Centers A number of both clinical • As described above, the only prospective ran- and laboratory studies have been reported from other centers domized clinical trial of pH strategy in which that have confrmed the advantages of the pH stat strategy for many patients underwent deep hypothermic pediatric bypass. We have seen no cases of persistent choreoathetosis since we 88 One strategy which makes little sense is to cool to deep adopted the pH stat strategy (Box 10. Alpha stat is associated with a greater cerebral metabolic rate Why Do Some Centers Continue to Use Alpha than the pH stat strategy that is obviously undesirable if cir- Stat or Hybrid Strategies for Congenital Cases? Continuing use of pH stat at hypo- Perfusionists who mainly work with adults are used to thermia allows a lesser rate of low fow bypass or greater using the alpha stat strategy. They may not be comfortable degree of hemodilution or longer safe duration of circulatory with the slightly greater complexity of the pH stat strategy. However, the hyperoxygenation achieved with ble oxygenator markedly decreased the number of gaseous modern oxygenators has the potential to aggravate ischemia- microemboli that could be detected. Interestingly, with either reperfusion injury during reperfusion through generation of 92–94 bubble or membrane oxygenators, temperature gradients oxygen free radicals, particularly in the heart.

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The response of exogenously administered epinephrine depends on the ratio of alpha- to beta-receptors in the individual tissue beds as well as to the dose of epinephrine given purchase genuine malegra fxt plus on line erectile dysfunction 14 year old. Activation of the β2-receptors in the vasculature of the skeletal muscles usually leads to a decrease in the systemic vascular resistance and the diastolic pressure generic malegra fxt plus 160 mg visa erectile dysfunction neurological causes. As the dose is progressively increased discount 160mg malegra fxt plus with amex erectile dysfunction lipitor, more prominent peripheral vasoconstriction is seen due to the activation of the α-receptors in other vascular beds (74). Renal blood flow is consistently decreased as vascular resistance in all segments of the renal vasculature increases (75). Epinephrine is often used as a strong inotrope in the support of the failing myocardium. During cardiopulmonary resuscitation, epinephrine is the vasopressor of choice since it has profound α- adrenergic stimulation that aids in maintaining the cerebral and coronary perfusion pressure during cardiovascular collapse (76). The American Heart Association recommended dose of epinephrine in children for bradycardia, asystolic or pulseless arrest is 0. Dopamine Dopamine is a naturally occurring catecholamine that is an immediate precursor of norepinephrine. Most of the functions of endogenously excreted dopamine are as a central neurotransmitter, though it has been found in the peripheral circulation as well. The cardiovascular effects of exogenously administered dopamine are due to the activation of a variety of receptors that have different affinity for the drug (80). However, “renal dose” dopamine has not been demonstrated to have direct beneficial effects in improving renal function (82). As the dose of the drug is increased, stimulation of the β1-receptors in the myocardium has inotropic and chronotropic effects (83). At these doses, dopamine causes an increase in cardiac output, decrease in pulmonary capillary wedge pressure, and there is usually a decrease in systemic vascular resistance with only slight changes in blood pressure. Total peripheral resistance is usually unchanged with low or intermediate doses of dopamine, due to vasodilatory action of dopamine on regional vascular beds. At higher doses (>10 mcg/kg/min), more α1-receptors are activated leading to a more intense peripheral vasoconstriction and an increase in vascular resistance. Dopamine causes release of norepinephrine from nerve endings; this also adds to its pharmacologic effect of adrenergic stimulation. The volume of distribution and the clearance of dopamine are highly variable, underscoring the principle of titrating this drug to effect in the individual patient (84). Dopamine in the dose range 5 to 15 mcg/kg/min is commonly used as an inotropic support to assist in the weaning from cardiopulmonary bypass, and in the early postoperative period. In recent years some practitioners have avoided dopamine because of its role as a neurotransmitter, which can cross the blood–brain barrier and is known to suppress pituitary function, particularly thyroid releasing hormone, in infants and children (85). This potential adverse effect is not seen with other natural or synthetic catecholamines (86). Dobutamine has not been shown to have any effect on the dopaminergic receptors or lead to the release of norepinephrine from nerve endings. The primary action of dobutamine is on β1-receptors with only a small effect on β2- or α1-receptors. Cardiac output is markedly enhanced and the left-sided filling pressures are decreased. This effect may be especially beneficial in treating patients with ventricular dysfunction. Comparison with newer inotropic drugs such as milrinone demonstrates similar improvements in stroke volume but a more profound decrease in left ventricular filling pressures and vascular resistance than with the phosphodiesterase inhibitors (88). Higher doses of dobutamine (>15 mcg/kg/min) can predispose to the development of atrial or ventricular arrhythmias. Because of the dual effects on the inotropic state of the heart and the vascular resistance, milrinone has been used extensively in the treatment of congestive heart failure, pulmonary hypertension, and postoperative low cardiac output. Peripheral vasodilation also ensues as a result of vascular smooth muscle relaxation. Hypotension and reflex tachycardia may result as a side effect of milrinone therapy. Milrinone is primarily renally excreted and higher bolus doses (50 to 75 mcg/kg) may show prolonged hemodynamic effects in patients with impaired renal function. Milrinone has also been suggested to have a higher volume of distribution and a faster clearance in infants and children as compared to adults (99). The dose recommended for milrinone therapy in patients with normal renal function is a bolus of 50 mcg/kg followed by an infusion of 0. Hypotension seen with a loading dose may be avoided by reducing or eliminating the loading dose and simply beginning the infusion, recognizing that therapeutic plasma levels will not be achieved for several hours. In a pharmacokinetic study of 16 neonates undergoing Norwood Stage I palliation, a loading dose of 100 mcg/kg into the bypass circuit at the start of rewarming provided therapeutic plasma concentrations, but an infusion of 0. Calcium The calcium ion is an integral part of the excitation–contraction coupling and impulse generation in myocardial cells and is a major determinant of vascular smooth muscle tone. Particularly in neonates, where the sarcoplasmic reticulum is not well developed, and the sequestration and release of calcium is thus inefficient, an adequate ionized calcium concentration is important to optimize myocardial contractility. Administration of calcium in the form of calcium chloride or calcium gluconate helps improve the inotropic function of the heart in the presence of hypocalcemia (102). Calcium functions primarily as a vasoconstrictor when the serum ionized calcium levels are normal. Moreover, increasing evidence suggests that elevated intracellular calcium levels are associated with cell death and injury during ischemia and reperfusion injury (104). A higher and more predictable amount of elemental calcium is available from the intravenous administration of calcium chloride than calcium gluconate or gluceptate (106). The routine use of calcium as a cardiotonic agent early after bypass in the presence of normocalcemia is not well supported. Indeed there is good evidence from resuscitation literature and in adults that boluses of calcium or supranormal levels of calcium are associated with adverse outcomes. Calcium administration is not recommended in bradyasystoles unless severe hypocalcemia or hyperkalemia coexists or if the arrest is secondary to calcium channel antagonist drugs (77,78). Chronotropes Isoproterenol Isoproterenol is a potent nonselective β-adrenergic agonist with only very minimal actions on α-receptors. Due to its vasodilatory β2-stimulatory actions as well as lack of α-receptor stimulation, isoproterenol leads to lowering of peripheral vascular resistance (73,108). An intravenous infusion of isoproterenol has more chronotropic than inotropic effect, as opposed to dopamine or dobutamine. Myocardial oxygen demands are greatly exacerbated by isoproterenol and this may exacerbate or induce ischemia (109). Higher doses of isoproterenol can be arrhythmogenic and may induce ventricular tachycardia or fibrillation. This agent is contraindicated in dynamic obstruction of the right or left outflow tracts. Isoproterenol is most commonly used as a temporary chronotropic agent in increasing the heart rate in patients with severe bradycardia or bradyarrhythmia (77,78) or early after cardiac transplantation. Vasoconstrictors Norepinephrine Norepinephrine is an endogenous catecholamine that is primarily released by the postganglionic adrenergic nerve endings. Besides being a major source of epinephrine, the adrenal medulla also contains norepinephrine in a smaller fraction (10% to 20%).

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A short segment of the distal end of the left fourth result in obstructive symptoms of both purchase 160 mg malegra fxt plus overnight delivery disease that causes erectile dysfunction. Nevertheless discount 160 mg malegra fxt plus with mastercard erectile dysfunction in young age, the arch persists as an aortic diverticulum generic 160 mg malegra fxt plus fast delivery diabetes erectile dysfunction wiki, which gives rise to mere presence of a ring does not guarantee that there will be the ligamentum. Note that the aortic diverticulum has been reported as the site of origin of aortic dissection. Also note that when there is mirror-image branching, if the ligamen- tum arteriosum arises from the innominate artery to pass to the origin of the left pulmonary artery rather than from the diverticulum of Kommerell this does not result in a vascular ring (Fig. Dominant Left Aortic Arch A dominant left aortic arch is extremely rare but should be recognized, because the best approach for surgical division is through a right thoracotomy. The ligamentum arteriosum contributes to secondary tracheoesophageal compression and should be divided together with the left anterior arch. Ao a double arch from the retroesophageal subclavian or ligament, based on the angulation of the esophageal impression. However, it is be through a left thoracotomy, which will be true in more an essential part of the workup for congenital tracheal stenosis. If a double aor- tic arch is present, it is important to be aware preoperatively Aortography which of the arches is dominant. In the majority of cases, it Expensive, invasive, and carrying additional risk for the will be the right aortic arch. Nevertheless, in cases of dou- patient, aortography is rarely justifed for the diagnosis of a ble patent arches it is useful to place pulse oximeter probes vascular ring. In addition, if there is an atretic segment, such on both hands and one foot so that temporary occlusion of a study will not be diagnostic. Because the point of division will be the or dysphagic symptoms are present, surgical division of the narrowest segment of the ring, there should be no pressure ring is indicated. Preoperatively the child should be given maxi- eral decubitus position, and a left posterolateral thoracotomy is performed that is more posterior than lateral. The chest is mal nutritional support as well as general respiratory care, entered through the fourth intercostal space, and the left lung including chest physiotherapy and appropriate treatment of is retracted anteriorly. Surgery should not be unduly delayed reveal a taut, ligamentous structure in the case of a ring with because of the presence of a respiratory infection, as division an atretic left arch. If a double arch is present, it is gener- of the ring, which allows more adequate clearing of respira- ally visible to some degree through the mediastinal pleura. The vagus nerve, giving off the left recurrent laryngeal nerve (which then passes around the ligamentum arteriosum), is a inDicAtions for AnD timing of surgEry useful landmark. The segment to be divided, if patent, should be controlled tory infections, dysphagia, refux, and failure to thrive. After division the vessel ends are oversewn with mild symptoms appearing for the frst time in the older infant a continuous Prolene suture. If the segment to be divided is may improve with time as the child grows, in which case clearly atretic, it suffces to doubly ligate the cord and divide it may be possible to defer surgery. After division the ends generally retract briskly, indicat- the risks of surgery are extremely small, the child should ing the tension with which the ring has been surrounding the undergo surgery within a reasonably short time from diagno- esophagus and trachea. The maximal delay until surgery should be determined tant that the ligamentum arteriosum should also be divided. There may be additional fbrous strands passing across the Vascular Rings, Slings, and Tracheal Anomalies 655 esophagus, and these should be divided. Final palpation in rEsults of surgEry the area should reveal complete relief of the taut band that Traditional Thoracotomy Approach was present previously. Between 1947 and 1992, 37 patients underwent traditional surgery for relief of tracheo- together with absorbable suture to the muscle layers, with esophageal obstruction caused by vascular rings. Of the 37 subcutaneous and subcuticular absorbable suture completing patients, 18 had a double aortic arch, 11 had a right aortic arch wound closure. In the rare case requiring approach through a with aberrant left subclavian, 4 had a left aortic arch with right thoracotomy, the same principles are applied. There was one early postop- Video-Assisted Technique of erative death and no late deaths. At long-term follow-up three Management of Vascular Ring patients had residual symptomatic tracheomalacia, one of This has become the method of choice for all vascular rings whom required right middle and lower lobectomy for recur- unless preoperative studies suggest that there is a patent rent pneumonia. A similar conclusion was drawn by Bakker position following single lumen endotracheal intubation. There was no operative mortality after tive feld achieved by retracting the infated left upper lobe 1959. The ring is dissected free from patients had preoperative or intraoperative bronchoscopy. The atretic segment The technique of operation shifted to a muscle-sparing left thoracotomy without routine chest drainage. Clips are patients with right aortic arch and a Kommerell diverticulum, placed and the ring and ligamentum are divided between clips. Of note, there was transferred to the left carotid artery as a primary pro- are limited thorascopic options to plicate the diverticulum of cedure. Primary reimplantation of the left subclavian artery Kommerell as one could in an open approach. Due to the thin- for right aortic arch with Kommerell’s diverticulum is also walled nature of the diverticulum, suture plication via a thora- 22 recommended by Shinkawa et al. Seventy-fve % (135/180) were free from direct vision and the wounds are closed with Steri-strips. Postoperative Management Results of Video-Assisted Division of Vascular Ring In the young infant with severe respiratory symptoms, there In 1995, Burke et al. Therefore, it should be anticipated that not all age of 5 months underwent the procedure. Four had a double respiratory symptoms will be relieved immediately; in fact, aortic arch with atretic left arch and four had a right arch it may be several months before the child is free of stridor. All eight However, there should be complete and immediate relief of patients had successful ring division with symptomatic relief any diffculty with feeding. In three patients a limited thoracotomy was 656 Comprehensive Surgical Management of Congenital Heart Disease, Second Edition performed to divide vascular structures. Similar results complete tracheal rings, that is, the posterior membranous were described by Koontz et al. The complete rings may be localized to the region where the sling passes around the tra- In a patient with a typical pulmonary artery sling the left chea, although often they extend for the entire length of the pulmonary artery arises from the right pulmonary artery trachea. Severe stenosis can involve the carina and extend for and passes leftward between the trachea and esophagus (Fig. The left pulmonary artery is pulmonary artery is probably not the more important mecha- often relatively hypoplastic and considerably smaller than the nism of tracheal stenosis as suggested by the following expe- right pulmonary artery, which itself appears larger than nor- rience. Several years ago we managed a child at Children’s mal and almost like a direct extension of the main pulmonary Hospital Boston with a pulmonary artery sling in which artery. The small size of the left pulmonary artery may help the sling passed around the trachea above a very high “pig RecurrentRecurrent laryngeal n. The procedure is best performed through a median sternotomy employing cardiopulmonary bypass.

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Additionally quality malegra fxt plus 160mg erectile dysfunction 19 years old, α-complementation requires only a small gene to be carried on the plasmid buy malegra fxt plus 160mg without prescription erectile dysfunction treatment chennai. In general malegra fxt plus 160 mg without prescription erectile dysfunction bangalore doctor, the stability of many plasmid vectors decreases as their size increases. Since the α-polypeptide is small, the total size of the vector is minimized, allowing it to carry a correspondingly larger insert. The choice of selectable marker usually rests with the type of cell that is being transformed. Some of the markers will only function against prokaryotes, while others have a broader spectrum of action. Some of the commonly used selectable markers are listed below, together with their mechanism of action. Over time the ampicillin in a culture medium or petri dish may be substantially destroyed by β-lactamase. When this occurs, selective pressure to maintain the plasmid is lost and cell populations can arise that lack the plasmid. Selective pressure will be maintained throughout the cell culture process to keep the plasmid containing the drug resistant gene. The Sh ble gene from the bacterium Streptoalloteichus hindus- tanus encodes a small protein that confers resistance to zeocin by binding to the antibiotic (Gatignol, Durand and Tiraby, 1988). Many of these will be discussed in later chapters of this book, but here I will list a number of examples to give the reader a flavour of the diversity of plasmid use. For example, plasmids for the cloning and expression of genes in the yeast Saccharomyces cerevisiae contain both replication origins and selectable markers for both E. High-level protein production could be driven from a strong promoter, while low-level production would be driven from weaker promoters. Levels of protein production may also be modulated by altering the copy number of the plasmid. For example, if you have cloned a gene, you might want to express the gene product at high levels in E. Genes are trans- ferred from the donor plasmid to the acceptor plasmid at the loxP sites using the Cre recombinase. These sites consist of two 13 bp inverted repeats separated by an 8 bp spacer region. The 8 bp spacer region in the loxP site has a defined orientation that forces recombination to occur in a precise direction and orientation. Acceptor plasmid contain a single loxPsiteand elements to which the target gene will become fused. Furthermore, if the coding sequence for the gene of interest is in frame with the upstream loxP site in the donor vector, it will automatically be in frame with all peptides designed in the acceptor vector. An alternative donor and acceptor plasmid system is based upon site-specific recombination reactions mediated by phage λ (Karimi, Inze and Depicker, 2002). The versatility of plasmids has lead to their widespread acceptance as the vectors of choice for many gene manipulation experiments. First, the efficiency at which the plasmid is transferred to a bacterial cell is very low. The viruses, more commonly called bacteriophages or simply phages, are able to infect other E. However, bacteria that had previously been exposed to phage λ, but had not undergone lysis, showed this remarkable property. Either cell lysis proceeds and newly synthesized phage particles will be released into the surrounding medium, or, alternatively, the phage can switch into a 3. An electron micrograph of λ phages that have been released upon bacterial lysis, and a diagrammatic representation of the overall structure of λ. In the laboratory, λ phage growth and replication is monitored on petri dishes (Figure 3. The mixture is poured onto the surface of a nutrient agar plate and incubated to allow bacterial growth. Such sites are observed on the plates as somewhat turbid plaques in the bacterial lawn. The genetics and molecular biology of bacteriophage λ have been extensively studied – for further information on λ, readers are directed to the excellent text by Mark Ptashne (Ptashne, 1992). The extreme 5 -and 3 -ends of the λ genome have 12 bases that are single-stranded – called the cohesive or cos ends. Functionally related genes of λ are generally clustered together on the λ genome, except for the two positive regulatory genes N and Q. Genes on the left-hand side of the conventional genome map code for head and tail proteins of the phage particle. The major advantage of λ basedvectorsoverplasmidsis the efficiency at which the phage can infect E. To understand how λ can be exploited as a vector, it is important to have a basic knowledge of the phage itself. Infection occurs as a result of the adsorption of the λ phage particle to the bacterial cell by binding to the maltose receptor. Eventually, bacterial cell lysis occurs and the newly formed phage are released into the surrounding medium. The mixture is then poured onto an already set agar plate where the top agar is allowed ◦ to solidify. Many of the genes required for the integration of λ into the host chromosome, or for new phage replication and assembly, are grouped together on the λ chromosome. A region of the genome that is not required for lytic growth is indicated frequently lysogenize. This behaviour makes sense, for in starved cells there will be less of the components necessary to make new phage particles. This transcription is subject to repression by the product of the cI gene and in a lysogen this repression is the basis of immunity to superinfection. The protein product of the cro gene builds up to a critical level and then stops early transcription. The product of the Q gene activates transcription, resulting in the production of the proteins required for the head and tail of the mature phage particle, and those required for bacterial cell lysis. Upon cell lysis, approximately 100 newly synthesized phage particles are released from a single infected bacterial cell. Two important developments, however, suggested that λ might be suitable as a cloning vector. Firstly it was determined that the gene products required for recombination could be removed from the λ genome and the lytic life cycle could still be completed and plaques would form. Several other basic strategies have been devised to identify λ phage recombinants. Screening of this type is technically difficult and requires a deal of skill on the part of the observer. Two different λ lysogens are used to produce the various components required for the packaging of λ particles.