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At pH 8 discount chloromycetin 500mg online medications used for fibromyalgia, both conjugated discount chloromycetin 250mg free shipping symptoms 11dpo, reductase unconjugated order chloromycetin 500mg with visa treatment anal fissure, and delta bilirubin react with the C generic chloromycetin 500 mg overnight delivery medicine 74. Using diﬀerent polarity modiﬁers enzyme, but at pH 4 only the conjugated form reacts. C Measurement of bilirubin concentration through Chemistry/Apply principles of basic laboratory the skin requires the use of multiple wavelengths procedures/Bilirubin/2 to correct for absorbance by melanin and other light-absorbing constituents of skin and blood. What is the principle of the transcutaneous than 100 wavelengths and multiple reﬂectance bilirubin assay? Multiwavelength reﬂectance photometry They can be used to identify neonates with D. To Clinical chemistry/Calculate/Solutions/2 calculate, multiply the percentage (as mL) by the 3. A biuret reagent requires preparation of a stock volume required (mL), then divide by 100 (mL). C To convert from milliequivalent per liter to milligrams stock solution are needed to prepare 100. B To calculate the volume of stock solution needed, divide the concentration of working standard by the concentration of stock standard, then multiply by the volume of working standard that is needed. C1 × V1 = C2 × V2, where C1 = concentration of stock standard V1 = volume of stock standard C2 = concentration of working standard V2 = volume of working standard 2000. B When zeros appear by themselves to the left of Clinical chemistry/Calculate/pH/2 the decimal point, they are not significant. How many signiﬁcant ﬁgures should be reported they are to the left of the decimal point and are when the pH of a 0. In laboratory practice, most analytes are reported Clinical chemistry/Calculate/Signiﬁcant ﬁgures/2 with two significant figures. B The Henderson–Hasselbalch equation can be used to determine the pH of a buﬀer containing a weak acid and a salt of the acid. A glycerol kinase method for triglyceride calls for a result for the patient’s serum before applying the serum blank in which normal saline is substituted ratiometric formula to calculate concentration. A The acceptable range for quality control results is deviations usually set at the 95% conﬁdence interval. This is Chemistry/Evaluate laboratory data to assess deﬁned as the range between –1. Chemistry/Evaluate laboratory data to assess validity/ If a control is assayed 100 times, 68 out of 100 results Accuracy of procedures/Quality control/1 would fall within +1 s and –1 s of the mean. Two consecutive controls greater than 2 s above 100 results fall within ±3 s of the mean. A Rejecting a run when three consecutive controls fall the mean between 1 and 2 s or when a trend of four increasing C. Four controls steadily increasing in value but less or decreasing control results occurs would lead to than ±1 s from the mean frequent rejection of valid analytical runs. One control above +1 s and the other below –1 s control limits are four consecutive controls above or from the mean below 1 s (41s) to detect a signiﬁcant shift, and a cusum result exceeding the ±2. When controls deviate in opposite directions, the diﬀerence should exceed 4s before the run is rejected. One of two controls within a run is above +2s Answers to Questions 20–24 and the other control is below –2s from the mean. The R4S rule is applied only to controls within a run Chemistry/Evaluate laboratory data to recognize (Level 1 – Level 2), never across runs or days. Two consecutive controls are both beyond –2s Ninety-ﬁve percent of the results fall within ±2s of the from the mean. B The R4s rule is applied to two control levels within Accuracy of procedures/Quality control/2 the same run. The R4s rule detects random error standard deviation from the mean (error due to poor precision). Two consecutive controls in the same run are control results either increase or decrease in the same each greater than ±4s from the mean direction; however, this is not cause for rejection until D. Trends are systematic errors consecutive controls (aﬀecting accuracy) linked to an unstable reagent, calibrator, or instrument condition. For example, loss Chemistry/Evaluate laboratory data to assess of volatile acid from a reagent causes a steady pH validity/Accuracy of procedures/Quality control/2 increase, preventing separation of analyte from 23. Recalibrate, then repeat controls followed by can be evaluated by repeating abnormal patient selected patient samples if quality control is samples. If the average diﬀerence between results acceptable before and after recalibration is > 2s, then all samples B. Prepare fresh standards and recalibrate Chemistry/Evaluate laboratory data to take corrective action according to predetermined criteria/Quality control/3 228 Chapter 5 | Clinical Chemistry 25. B Data between ±2 and ±3s must be included in current target limits calculations of the next month’s acceptable range. Using control results from all shifts on which the “out-of-control” situations a frequent occurrence. A Although calcium has the lowest s, it represents the Chemistry/Apply principles of laboratory assay with poorest precision. Relative precision operations/Quality control/2 between diﬀerent analytes or diﬀerent levels of the same analyte must be evaluated by the coeﬃcient 26. For example, when comparing the precision of the level 1 control to the level 2 control, A. The method mean for comparison of precision and accuracy among level 1 is at the center of the y axis and mean for level laboratories? Which plot will give the earliest indication of a Answers to Questions 30–31 shift or trend? Results are out problems/Quality control/2 of control when the slope exceeds 45° or a decision 31. Te matrix is similar to the specimens being dynamic linear range of the assay, and can be used tested for multiple analytes. Te concentration of analytes reﬂects the clinical is determined from replicate assays by the user‘s range method, not the “true” concentration of the analyte. Analyte concentration must be independent of Out-of-control results are linked to analytic the method of assay performance rather than to the inherent accuracy Chemistry/Apply principles of basic laboratory of the method. Kurtosis temperature, evaporation, light exposure, and Chemistry/Evaluate laboratory data to recognize bacterial contamination. Day 9 plotting control data is that trends can be identiﬁed Chemistry/Evaluate laboratory data to recognize before results are out of control and patient data problems/Quality control/3 must be rejected. In this case, corrective steps should have been implemented by day 7 to avoid the delay 34. Referring to the Levy–Jennings chart, what and expense associated with having to repeat the analytical error is present during the second analysis of patient samples. Kurtosis in the assay conditions that aﬀect the accuracy of all results, such as a change in the concentration of the Chemistry/Evaluate laboratory data to recognize calibrator; change in reagent; a new lot of reagent problems/Quality control/3 that diﬀers in composition; or improper temperature 35. What is the ﬁrst day in the second half of the setting, wavelength, or sample volume. Day 19 This means that four consecutive controls are greater Chemistry/Evaluate laboratory data to recognize than ±1s from the mean. D An R4s error is deﬁned as the algebraic diﬀerence +2s between two controls within the same run.
Although this is generally disadvantageous for drug delivery generic chloromycetin 250mg line medications 230, first-pass metabolism can be beneficial for prodrugs discount chloromycetin 250mg fast delivery treatment jokes, which rely on drug metabolism for activation purchase chloromycetin 250mg mastercard treatment ingrown hair. Drugs that structurally resemble nutrients such as polypeptides chloromycetin 500 mg online medications used to treat adhd, nucleotides, or fatty acids may be especially susceptible to enzymatic degradation. For example, the proteolytic enzymes chymotrypsin and trypsin can degrade insulin and other peptide drugs. In the case of insulin, proteolysis was shown to be reduced by the coadmmistration of carbopol polymers at 1% and 4% (w/v%), which presumably shifted the intestinal pH away from the optimal pH for proteolytic degradation. Drugs such as erythromycin, penicillin, and omeprazole are unstable in acidic media, and will therefore degrade and provide lower effective doses depending on the gastric pH, drug solubility, and residence time of the dosage form in the stomach. Thus, hydrophobic substrate molecules that enter the membrane lipid bilayer from the lumen will be extracted directly back to the extracelluar medium by the P-glycoprotein, prior to reaching the cell cytoplasm. An alternative model proposes that substrate efflux through the pump (at low substrate concentration) occurs via a four-step mechanism. The drug substrate is bound to P-glycoprotein on the cytoplasmic side of the cell membrane. There is a high level of expression of P-gp in the epithelial cells of the small intestine. Compounds that have been found to be substrates exhibit a wide range of chemical structures. However, they tend to be lipophilic and, for some, cationic, such as anthracyclines, vinca alkaloids, cyclosporin, etoposide, and celiprolol. It has been shown that taxol, an anti-microtubule anticancer drug, was not absorbed after oral administration in pre-clinical trials. This can probably be attributed to P-gp, since the flux from the 140 basolateral to the apical side was 4–10 times greater than in the opposite direction. Thus, P-gp may play an important role in determining the oral bioavailability of certain drugs. Food may reduce the rate or extent of absorption by a number of mechanisms: • By slowing down gastric emptying rate, which is a particularly important effect for compounds unstable in gastric fluids and for dosage forms designed to release drug slowly. Enzymes present in these fluids may deactivate a drug moiety; similarly, increased acid secretion provoked by the presence of food may cause increased degradation of acid-labile compounds. The deleterious effects of food on drug absorption have prompted the use of dietary strategies in order to improve oral bioavailability. For example, the drug L-dopa, used in the treatment of Parkinson’s disease, is absorbed via a stereospecific, saturable active transport mechanism shared by large neutral amino acids such as phenylalanine and tyrosine. The breakdown products of dietary proteins can compete with L-dopa for this active transport mechanism, thereby reducing its oral bioavailability. Taking L-dopa at least 30 min before eating and controlling dietary protein has been shown to improve L-dopa treatment in Parkinson’s disease. A further example is the avoidance of milk 2 h prior to taking preparations containing tetracyclines, as these drugs chelate calcium ions in milk, forming a poorly absorbable complex. Interestingly, the presence of food may favor drug absorption in other situations. The positive effect of food on the absorption of this drug was also observed with Eudragit S100 nanoparticles. The administration of a 150 mg diclofenac hydrogel-based capsule dose within 30 min following a standardized breakfast was shown to minimally affect the bioavailability of dicolfenac relative to administration under fasted conditions. The insoluble fraction forms 141 a semi-impermeant layer, which, in conjunction with bicarbonates (secreted by gastric cells at the surface and in gastric pits), protects underlying cells from damage by gastric acid. Studies have shown that gastrointestinal mucus presents a physical barrier to the diffusion of small molecules such as urea, benzoic acid, antipyrine, l-phenylalanine and warfarin as well as to large protein molecules. Similarly, the passive absorption of testosterone was shown to be doubled upon ridding the intestinal epithelial cells of the overlying mucus layer. However, the situation regarding the effect of mucus on oral bioavailability is a complex one; for example, it has been shown that drug binding to the mucosal surface is essential to the absorption of barbituric acid derivatives from the rat small intestine. Gender Gastric acid secretion is greater in men than in women, whereas gastric emptying time is slower in women. Enzyme expression is also different between men and women; for example, sex-related cytochrome P-450 isozymes and glucuronidation enzymes are more abundant in men. However, in general, gender differences are small and insufficient to warrant a modification in dosage regiments. Pregnancy results in reduced gastric acid secretion, increased intestinal motility, increased plasma volume, decreased plasma drug binding and also an additional pharmacokinetic compartment. These altered pharmacokinetic factors may require modifications in the dosage regimen for certain drugs. Race Racial differences in oral drug bioavailability are known to exist and may be due to environmental, dietary or genetic differences. These differences are becoming increasingly important in therapeutics, due to both the increasingly international nature of drug development and use, and also the multi-racial nature of the population of many countries. The hydroxylation defect for debrisoquine also applies to the oxidative metabolim of codeine, metoprolol, and perphenazine. The clinical conse-qunces of polymorphic oxidation have not been examined in great detail. Obviously, the small percentage of the population who are poor metabolizers may be at considerable risk of adverse effects from the usual doses of many drugs. Age Few pharmacokinetic studies are carried out beyond the range of 28–40 years and, consequently, there are few data on oral bioavailability for extremes of age. Gastric fluid is less acidic in newborns than in adults, which can affect the absorption of ionizable and acid-labile drugs. Decreased enzymatic activity, including hepatic first-pass metabolism, is associated with the elderly, which may result in an increased oral bioavailabiliy for drugs subject to the first- pass effect. The effect of the shunt is to increase the presistence of the drug in the body and, provided the concentrations of the drug at its sites of action are sufficiently high, to prolong its duration of action. It is important to remember that although a drug molecule may be predominantly absorbed via one particular route/mechanism, it is also likely that suboptimal transport will occur via alternative routes and mechanisms. Diffusion is driven by a concentration gradient and is inversely related to molecular weight. The junctional complexes begin immediately below the luminal surface and are made up of three components (Section 1. Thus only small hydrophilic molecules, such as, for example, mannitol, are capable of squeezing through the junctional complexes to be absorbed via the paracellular route. The rate of absorption is governed by Fick’s Law and is determined by the physicochemical properties of the drug as well as the concentration gradient across the cells (Section 1. Carrier-mediated transport Amino acid transporters, oligopeptide transporters, glucose transporters, lactic acid transporters, monocarboxylic acid transporters, phosphate transporters, bile acid transporters and other transporters present on the apical membrane of the epithelial cells serve as carriers to facilitate nutrient absorption by the intestine. Drug moieties possessing similar structures to nutrients that are absorbed by such carriers may also be absorbed in this manner. Endocytic processes Considerable evidence has accumulated indicating that macromolecules and microparticulates can be taken up by the intestinal enterocytes, generally via pinocytosis. For example, studies have shown that receptor-mediated endocytosis via enterocytes is a major pathway for the intemalization of certain antisense oligonucleotides. In contrast, endocytic uptake of macromolecules and microparticles is carried out extensively by the M cells of the 144 Peyer’s patches. Transcellular shuttling through the M cells to the underlying Peyer’s patch may involve an adsorptive and/or receptor-mediated process, with membrane-bound vacuoles or vacuoles already present in the apical cytoplasm of the cells (see below, Section 6. Therefore, they are ionized to a certain extent, determined by their pKa and the pH of the biological fluid in which they are dissolved; the extent of ionization can be quantified by the Henderson-Hasselbalch Equation (see Section 1.
In the younger cheap 250mg chloromycetin amex medicine rock, more athletic popu- lation generic 500mg chloromycetin with amex symptoms you need glasses, glenohumeral dislocation frequently can lead to glenohumeral Figure 33 quality chloromycetin 250 mg medications via g-tube. The risk of redislocation of the glenohumeral joint after a primary traumatic dislocation ranges from 70% to 90% generic chloromycetin 250 mg fast delivery medications multiple sclerosis. The subse- quent dislocations usually require less trauma than the index disloca- tion. In many cases, recurrent instability of glenohumeral joint requires surgical intervention. The treatment usually consists of repairing the anterior inferior capsule and the inferior glenohumeral ligament complex to the rim of the glenoid. This occurs when patients fall on the lateral side of their upper arm or run into a hard object, such as an outﬁeld wall in baseball. However, the clavicle is ﬁxed rigidly by the sternoclavicular joint and really does not rotate signiﬁcantly. However, in more signiﬁcant injuries, dis- ruption of the ligaments is accompanied by penetration of the delto- trapezial fascia by the distal clavicle. In this case, the distal clavicle is in a subcutaneous position and will lead to chronic pain. The usual treatment is for repair or reconstruction of the coracoclavicular ligaments and repair of the del- totrapezial fascia. Although there is initial dysfunction of the shoulder, the scapula body heals, and good return of function is expected. The only fracture of the scapula that may require surgical intervention is an intraarticular fracture of the glenoid that involves greater than 30% to 40% of the articular surface with a signiﬁcant step-off on the joint surface, or a fracture of the glenoid neck in conjunction with a fracture of the clavicular shaft that allows for medial migration of the shoulder. Since this portion of the bone is in a relatively subcutaneous position, deformities are visually obvious. This injury can be treated with a sling or a ﬁgure-of-eight strap, which attempts to retract the shoulders and to help align the fracture fragments. However, the ﬁgure-of-eight strap is difﬁcult to wear and often not well tolerated by patients. Restoration of essentially normal shoulder function is expected as clavicle fractures heal. Due to the osteopenic condition of their bone, the proxi- mal humerus is susceptible to fracture as the result of a fall onto the 604 C. In the majority of these injuries, the fractures are displaced minimally and often heal uneventfully, although shoulder stiffness or adhesive capsulitis can be a complicating factor during the recovery. Usually, in high-energy injuries to the proximal humerus, displacement occurs as a result of the fracture. In these cases, the fracture fragments usually are deter- mined by muscular attachment. The lesser tuberosity, which serves as a subscapu- laris attachment, becomes another. The articular surface of the humeral head is an individual fracture fragment and can be dislocated in an anterior or posterior direction. The junction between the humeral shaft and the humeral neck is another commonly involved fracture site. In cases in which there is signiﬁcant displacement of the tuberosity fragments, surgical inter- vention is indicated to restore glenohumeral function. In cases in which the articular surface of the humeral head is displaced in conjunction with the tuberosity and shaft fractures or in the case where the articu- lar surface is dislocated, it is difﬁcult to obtain adequate healing even with surgical intervention. Fracture of the humeral shaft usually occurs as a result of a twist- ing injury to the upper arm. Spiral fractures of the humeral shaft in the pediatric population can be a sign of physical abuse. It is impor- tant to assess the integrity of the radial nerve in conjunction with humeral shaft fractures, especially if the fracture has occurred between the junction of the middle and distal thirds of the humeral shaft. In the majority of cases of humeral shaft fracture with radial nerve injury, the radial nerve function returns spontaneously, and surgical exploration of the nerve rarely is indicated unless the fracture is an open fracture and surgical treatment is instituted for the prevention of infection. Elbow and Forearm The skeletal anatomy of the elbow consists of the humerus, ulna, and radius. The joint between the radial head and the capitellum allows for rotation about the long axis of the radius, thus allowing the pronation and supination of the forearm. Muscle strain injuries around the elbow are common, since the muscles around the elbow tend to set the wrist and forearm into a posi- tion of power for daily activities. Avulsion of the biceps tendon from its attachment on the radial tuberosity has a dramatic presentation. Although some patients believe that this injury leads to a loss of elbow ﬂexion strength, this is not the case. Patients who avulse their biceps from its distal insertion actually note a loss in forearm supination strength. In the younger patient, especially in those involved in stren- uous manual labor or those concerned with body symmetry, surgical reattachment usually is indicated. In the older patient, nonoperative treatment consisting of range of motion and strengthening usually pro- vides an excellent functional outcome, and surgery can be avoided. Triceps avulsions from the olecranon are much rarer and almost always require surgical treatment to restore elbow extension strength. Dislocations of the elbow joint almost always occur as a result of a fall onto an extended arm. As the elbow is forced into hyperextension, the joint dislocates such that the proximal ulna and the radial head lie posterior to the distal humerus. When evaluating a patient with an elbow dislocation, a complete neurovascular examination should be performed prior to reduction, since the brachial artery and the median, ulnar, and radial nerves all pass in close proximity to the elbow joint and can be injured at the time of the dislocation. Associated fractures of the radial head are detected on radiographic evaluation of the dis- location. Once the elbow joint has been reduced, a repeat neurovascu- lar examination should be performed, particularly to ensure no nerve entrapment has occurred during the reduction maneuver. As opposed to the shoulder, the elbow joint tends to be stable after dislocation, and the risk of recurrent dislocation is very low. Posttraumatic contractures are not uncommon and can be prevented with early motion and rehabilitation. Fractures about the elbow usually are the result of a fall with a direct blow to the elbow or a fall onto the outstretched hand. Fractures of the distal humerus involving the articular surface or the supracondylar region tend to be high-energy injuries with extensive comminution (Fig. Fractures of the proximal ulna, referred to as olecranon fractures, result in disruption of the elbow extensor mechanism, and these require surgical internal ﬁxation if displaced. Fractures of the radial head tend to occur as a result of a fall onto on outstretched arm. In general, these tend to be lower energy injuries and often have minimal displacement. The primary treatment of these is early motion to prevent posttraumatic contracture. However, if the radial head frag- ment is displaced severely and results in mechanical block to full motion, the fragment may need to be reduced and ﬁxed or excised sur- gically. A Monteggia fracture is a fracture of the proximal ulna with a dislocation of the radial head. Anatomic reduction of the ulnar shaft fracture almost always results in reduction of the radial head with good stability. Fractures of the shaft of the radius and ulna occur as a result of a direct blow to the forearm or a fall onto an outstretched hand.
Based on the morphology and continuity of the endothelial layer cheap chloromycetin 500mg otc symptoms questionnaire, capillary endothelium can be divided into three categories: continuous generic 250 mg chloromycetin visa treatment zoster, fenestrated chloromycetin 500mg with amex medicine misuse definition, and discontinuous endothelium (see Section 5 purchase 250 mg chloromycetin with mastercard medications and grapefruit juice. The continuous capillaries are found in skeletal, cardiac, and smooth muscles, as well as in lung, skin, subcutaneous and mucous membranes. The endothelial layer of brain microvasculature is the tightest endothelium, with no fenestrations. Capillaries with fenestrated endothelia and a continuous basement membrane are generally found in the kidney, small intestine and salivary glands. Most of these capillaries have diaphragmed fenestrae, which are circular openings of 40–60 nm in diameter. The discontinuous capillaries, also known as sinusoidal capillaries, are common in the liver, spleen, and bone marrow. These capillaries show large interendothelial junctions (fenestrations up to 150 nm). Highly phagocytic Kupffer cells line the sinusoids of the liver, and those of the bone marrow by flattened, phagocytic reticuloendothelial cells. In the spleen, the endothelial cells contain a large number of pinocytic vesicles (up to 100 nm in diameter). Due to their large molecular weight (> 1,000 kDa) and hydrodynamic diameter in aqueous suspension of 100 nm, plasmids extravasate poorly via continuous capillaries because of tight junctions between the cells. However, plasmids can easily extravasate to sinusoidal capillaries of liver and spleen. Formulating plasmids into unimeric particles of 20–40 nm in diameter may enhance extravasation of plasmids across continuous and fenestrated capillaries. The (patho)physiology and microanatomy of tumors is significantly different from normal tissues (see Section 5. A tumor contains vessels recruited from the pre-existing network and vessels resulting from angiogenic response induced by cancer cells. There is a considerable variation in the cellular composition, basement membranes and in the size of the interendothelial cell fenestrations. Tumor interstitium is characterized by large interstitial volume and high diffusion rate. Sven Frøkjaer, Lona Christrup and Povl Krogsgaard-Larsen; Munksgaard, Copenhagen, 1998, pp. Tumor accumulation of plasmid could result from the enhanced permeability of the tumor vasculature, combined with their reduced clearance from the tumor due to the absence of the lymphatic system. Pharmacokinetic analysis of in vivo disposition profiles of radiolabeled plasmid provides useful information on the overall distribution characteristics of systemically administered plasmids, with one critical limitation. The plasma half-life of plasmid is less than 10 min, and hence tissue distribution and pharmacokinetic parameters of plasmid calculated on the basis of total radioactivity are not valid at longer time points. Thus, polymerase chain reaction and Southern-blot analysis are required to establish the time at which the radiolabel is no longer an index of plasmid distribution. The deposition of plasmids after systemic administration is restricted to the intravascular space due to its low microvascular permeability in most organs with continuous capillary bed. Some organs with fenestrated capillaries, such as liver, spleen, and bone marrow, provide some opportunities for extravasation of plasmids. Intravenously injected plasmids initially perfuse the pulmonary vascular beds, maximizing the 347 Figure 14. Reproduced with permission from: Biodistribution and gene expression of plasmid/lipid complexes after systemic administeration, Mahato R. Southern-blot analysis of blood showed the rapid degradation of plasmid, with a half-life of less than 5 min for intact plasmid, and was no longer detectable at 1 hr postinjection. By Southern-blot analysis, there was no detectable plasmid in the brain, large intestine, small intestine, or gonads at the 1-hr timepoint. Southern blot analysis also demonstrated that plasmid remained in the liver, spleen, lung, marrow, and muscle, although at diminished levels, up to 24 hr postinjection. The plasma membrane is the next obstacle to be overcome in delivering genes into a cell. Gene delivery systems rely on binding to cell surface molecules, either specific, non- specific or both, prior to cellular internalization. The surface bound material usually gains entry into the cell either by endocytosis or membrane fusion. The schematic representation of the process of gene delivery and expression is shown in Figure 14. Gene delivery systems can distribute plasmids to the desired target cells, after which the plasmid is internalized into the cell by a number of mechanisms, such as adsorptive endocytosis, receptor-mediated endocytosis, micropinocytosis, caveolae-mediated endocytosis and phagocytosis (see Section 1. The intracellular fate of plasmids depends on the means by which they are internalized and translocated to the cytoplasms and then to the nucleus. Extacellular environment → tissue targetability → cellular uptake → intracellular trafficking → nuclear entry → gene expression The transition from coated vesicle to early endosome is accompanied by acidification of the vesicular lumen that continues into the late endosomal and lysosomal compartments, reaching a final pH in the perinuclear lysosome of approximately 4. Such acidification associated with endosome maturation provides the means by which certain viruses gain access to the cytosol. Acid-induced conformational changes in the viral proteins trigger translocation across the endosomal membrane via a fusion process. By taking advantage of the endosomal acidification, pH-sensitive liposomes, adenovirus and endosomolytic peptides have been used to facilitate the release of plasmids into the cytoplasm prior to lysosomal degradation. Non-clathrin-coated pit internalization can occur through smooth imagination of 150–300 nm vesicles or via potocytosis. This pathway has been shown to be involved in the transport of folate and other small molecules into the cytoplasm. Plasmids are taken up by muscles through the T-tubules system and caveolae via potocytosis. Muscle cells appear to take up plasmids through the T-tubule system and caveolae via potocytosis. Apart from coated or uncoated pit pathways, cells may also take up plasmid/cationic carrier complexes via plasma membrane destabilization. Particles greater than 200 nm in diameter are not 350 efficiently taken up by endocytosis, but cells may also take up some larger plasmid/cationic carrier complexes via phagocytosis. Plasmid/cationic carrier complexes have been proposed to internalize into the endosome and initiate the destabilization of endosomal membranes. This destabilization would induce diffusion of anionic lipids from the external layer of the endosomal membrane into the complexes and form charge neutralized ion pairs with the cationic lipids. Destabilization and/or fusion of the complex with the plasma membrane would permit the same anionic lipids to diffuse to the surface, as would fusion with the endosomal membrane. Transfection efficiency is dependent on mitotic activity, as cells prevented from going into mitosis after transfection express transgenes much less efficiently than proliferating cells. In search for an explanation, the transport of plasmids across the nuclear membranes has been studied. Plasmids injected into the cytoplasm of quiescent human fibroblasts are not expressed, in contrast to plasmid injected into the nucleus. This has been found to be true for the cationic lipid-based systems; as plasmid injected into the cytoplasm of Xenopus oocytes is not expressed, unlike that injected into the nucleus, it must be concluded that the plasmid must dissociate from the cationic lipids before entering into the nucleus. A fundamental limitation to gene expression using most of the gene delivery systems is the inability of plasmid in the cytoplasm to migrate into the nucleus. Microtubules and actin filaments have been proposed to be involved in intracellular trafficking of macromolecules, including plasmids. These cytoskeletal components maintain intracellular distribution of organelles and facilitate trafficking between organelles. Motor proteins, motor protein receptors, or the relevant peptide sequences may be conjugated to or complexed with plasmid.
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