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Biomedical subjects

P Alexander

Publications and source records attributed to P Alexander.

At least 19 recordsLinked to original sources
PubMed1992

Multidomain binding of transforming growth factor alpha to the epidermal growth factor receptor.

Solubilized epidermal growth factor receptor (EGF-R) has been used in an extension of the Geysen epitope mapping protocol in order to provide additional insight into the amino acid residues in human transforming growth factor alpha (hTGF alpha) which are critical to recognition and binding. Overlapping heptapeptides which encompassed the 50 amino acid primary sequence of hTGF alpha were synthesized on a polyethylene solid phase, and the amount of detergent-solubilized EGF-R bound to each peptide was measured using ELISA. EGF-R appeared to bind reproducibly to four heptapeptides cognate to sequences in both the N- and C-domains of hTGF alpha (residues 22-28, 28-34, 36-42, and 44-50). Visualization of these four regions on three-dimensional solution phase structures of hTGF alpha, derived from 1H NMR measurements [Kline, T.-P., Brown, F.K., Brown, S.C., Jeffs, P.W., Kopple, K.D., & Mueller, L. (1990) Biochemistry 29, 7805-7813], indicated that the peptide segments are located on a single face of the protein and suggested the presence of a potential receptor binding cavity. If peptide segments within both the N- and C-domains of hTGF alpha are involved in binding to EGF-R, then this has direct consequences for possible molecular mechanisms by which receptor activation might take place. For example, the observed conformational flexibility in the six NMR-derived hTGF alpha structures due to variations in the main-chain torsion angles of Val-33, in combination with the involvement of residues from both domains in the proposed binding cavity, may imply that receptor activation results from interdomain reorientation in the protein ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Kinetic analysis of folding and unfolding the 56 amino acid IgG-binding domain of streptococcal protein G.

The 56 amino acid B domain of protein G (GB) is a stable globular folding unit with no disulfide cross-links. The physical properties of GB offer extraordinary flexibility for evaluating the energetics of the folding reaction. The protein is monomeric and very soluble in both folded and unfolded forms. The folding reaction has been previously examined by differential scanning calorimetry (Alexander et al., 1992) and found to exhibit two-state unfolding behavior over a wide pH range with an unfolding transition near 90 degrees C (GB1) at neutral pH. Here, the kinetics of folding and unfolding two naturally occurring versions of GB have been measured using stopped-flow mixing methods and analyzed according to transition-state theory. GB contains no prolines, and the kinetics of folding and unfolding can be fit to a single, first-order rate constant over the temperature range of 5-35 degrees C. The major thermodynamic changes going from the unfolded state to the transition state are (1) a large decrease in heat capacity (delta Cp), indicating that the transition state is compact and solvent inaccessible relative to the unfolded state; (2) a large loss of entropy; and (3) a small increase in enthalpy. The most surprising feature of the folding of GB compared to that of previously studied proteins is that its folding approximates a rapid diffusion controlled process with little increase in enthalpy going from the unfolded to the transition state.

Amino Acid Sequence↗
PubMed1992

Energetics of folding subtilisin BPN'.

Subtilisin is an unusual example of a monomeric protein with a substantial kinetic barrier to folding and unfolding. Here we document for the first time the in vitro folding of the mature form of subtilisin. Subtilisin was modified by site-directed mutagenesis to be proteolytically inactive, allowing the impediments to folding to be systematically examined. First, the thermodynamics and kinetics of calcium binding to the high-affinity calcium A-site have been measured by microcalorimetry and fluorescence spectroscopy. Binding is an enthalpically driven process with an association constant (Ka) equal to 7 x 10(6) M-1. Furthermore, the kinetic barrier to calcium removal from the A-site (23 kcal/mol) is substantially larger than the standard free energy of binding (9.3 kcal/mol). The kinetics of calcium dissociation from subtilisin (e.g., in excess EDTA) are accordingly very slow (t1/2 = 1.3 h at 25 degrees C). Second, to measure the kinetics of subtilisin folding independent of calcium binding, the high-affinity calcium binding site was deleted from the protein. At low ionic strength (I = 0.01) refolding of this mutant requires several days. The folding rate is accelerated almost 100-fold by a 10-fold increase in ionic strength, indicating that part of the free energy of activation may be electrostatic. At relatively high ionic strength (I = 0.5) refolding of the mutant subtilisin is complete in less than 1 h at 25 degrees C. We suggest that part of the electrostatic contribution to the activation free energy for folding subtilisin is related to the highly charged region of the protein comprising the weak ion binding site (site B).(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus↗
PubMed1992

Thermodynamic analysis of the folding of the streptococcal protein G IgG-binding domains B1 and B2: why small proteins tend to have high denaturation temperatures.

We have cloned, expressed, and characterized two naturally occurring variations of the IgG-binding domain of streptococcal protein G. The domain is a stable cooperative folding unit of 56 amino acids, which maintains a unique folded structure without disulfide cross-links or tight ligand binding. We have studied the thermodynamics of the unfolding reaction for the two versions of this domain, designated B1 and B2, which differ by six amino acids. They have denaturation temperatures of 87.5 degrees C and 79.4 degrees C, respectively at pH 5.4, as determined by differential scanning calorimetry. Thermodynamic state functions for the unfolding reaction (delta G, delta H, delta S, and delta Cp) have been determined and reveal several interesting insights into the behavior of very small proteins. First, though the B1 domain has a heat denaturation point close to 90 degrees C, it is not unusually stable at physiologically relevant temperatures (delta G = 25 kJ/mol at 37 degrees C). This behavior occurs because the stability profile (delta G vs temperature) is flat and shallow due to the small delta S and delta Cp for unfolding. Related to this point is the second observation that small changes in the free energy of unfolding of the B-domain due to mutation or change in solvent conditions lead to large shifts in the heat denaturation temperature. Third, the magnitude and relative contributions of hydrophobic vs nonhydrophobic forces (per amino acid residue) to the total free energy of folding of the B-domain are remarkably typical of other globular proteins of much larger size.

Bacterial Proteins↗
PubMed1992

Sequence-specific 1H NMR assignments and secondary structure of the streptococcal protein G B2-domain.

Two-dimensional NMR spectroscopy has been used to obtain sequence-specific 1H NMR assignments for the IgG-binding B2-domain of streptococcal protein G. Secondary structure elements were identified from analysis of characteristic backbone-backbone NOE patterns and amide proton exchange data. The B2-domain contains a four-stranded beta-sheet region in which the two inner strands form a parallel beta-sheet with each other and antiparallel beta-sheets with the outer strands. The outer strands are connected via a 16-residue alpha-helix and short loops on both ends of the helix. The alpha-helix and beta-sheet structures contain well-defined polar and apolar sides, and numerous long-range NOEs from the apolar helix to apolar sheet regions were used to derive a model for the global fold of the B2-domain. While the overall fold is similar to that obtained for B1-type domains, differences in amide proton exchange rates and hydrophobic packing are observed.

Amino Acid Sequence↗
PubMed1991

Importance of the omentum in the development of intra-abdominal metastases.

Areas of trauma are preferred sites for metastatic tumour growth. In extensive intra-abdominal tumour recurrence the omentum is invariably involved. The importance of the omentum in the development of tumours at sites of intra-abdominal trauma has been investigated. Colonic anastomoses were performed in Hooded-Lister rats with and without omentectomy. Animals received intraluminal or intraperitoneal injections of a syngeneic tumour. With intraluminal injection, tumour occurred at the anastomosis and in the omentum in 38 and 43 per cent of animals respectively but following omentectomy the values were 14 and 9 per cent (omental remnant). With intraperitoneal administration tumour occurred in 53 per cent at the anastomosis and in 79 per cent in the omentum compared with 16 and 29 per cent (in omental remnant) following omentectomy. In this model a reduced ability of an anastomosis to support 'seeded' tumour following removal of the omentum is demonstrated and the development of local recurrence from spilled tumour cells during operation may be enhanced by, or be dependent on, the proximity of the omentum.

Anastomosis, Surgical↗
PubMed1991

Intrahepatic cholestasis induced by amoxicillin/clavulanic acid (Augmentin): a report on two cases.

Two patients who developed a severe but reversible cholestatic jaundice during and after treatment with an amoxicillin/clavulanic acid preparation (Augmentin) are described. The diagnosis of drug-induced intrahepatic cholestasis was based on the clinical course, biochemical pattern and on histological findings. Since amoxicillin alone does not induce liver injury, the combination of amoxicillin and clavulanic acid or clavulanic acid alone could be responsible for this adverse reaction, probably through a metabolic idiosyncrasy or a hypersensitivity mechanism. Although more than 100 cases of hepatotoxic reactions have been reported to the pharmaceutical company (Beecham Laboratories) and several reports have been published, the incidence of hepatotoxicity of this antibiotic is relatively rare, since this antibiotic has been widely used. However, recognition of this benign and reversible cholestatic syndrome is important to avoid unnecessary, expensive and invasive diagnostic procedures.

Aged↗
PubMed1990

Blood transfusion and recurrence of colorectal cancer: the role of platelet derived growth factors.

Efforts to explain the possible effects of blood transfusion on the recurrence of colorectal cancer have been based entirely on the immunosuppressive effects of blood transfusion. However, the relationship between solid tumour development and the immune system is inconclusive. We have investigated an alternative mechanism involving the potential role of growth factors in this phenomenon. Using a human fibroblast: [125I]deoxyuridine uptake mitogenesis assay, the relative amounts of growth factor in the plasma of stored blood were measured. There was a progressive increase in mitogenesis from day 0 (n = 6) to day 28 (n = 6; P less than 0.001, Mann-Whitney U test). The effect of growth factors on the development of liver and intraperitoneal metastases was studied in Hooded Lister rats. Following an intraportal injection of 10(5) MC28 tumour cells, the experimental group (n = 25) received 2 ml of syngeneic serum intravenously for 4 days. Likewise, colonic anastomoses were performed on omentectomized rats and the peritoneal cavity seeded with 10(3) cells. The experimental groups (n = 20) received either 2 ml serum intravenously repeatedly or 3 ml serum intraperitoneally (n = 19). There was no significant increase in liver metastases or peritoneal disease following intravenous infusion of serum but serum delivered intraperitoneally resulted in a significant increase in tumour from 22 per cent in the controls to 89 per cent in the study group (P less than 0.01). Growth factors released from platelets following blood loss into the peritoneal cavity may be important in enhancing local recurrence of colorectal cancer.

Anastomosis, Surgical↗
PubMed1990

Urinary epidermal growth factor (hEGF) levels in patients with carcinomas of the breast, colon and rectum.

A specific two-site ELISA for human epidermal growth factor (hEGF) has been used to measure urinary hEGF/creatinine ratios in 30 normal subjects, 30 hospital in-patients with breast cancer and 30 hospital in-patients with colonic or rectal cancer. There was no significant difference between patients with breast cancer and controls. Although a statistically significant difference between patients with colorectal cancer and controls was observed, the biological significance of this observation is doubtful. No clear effect of the presence of breast or colorectal carcinoma on the urinary excretion of hEGF has been observed.

Adult↗
PubMed1990

Coagulant and fibrinolytic activities of a metastasising and non-metastasising tumour line.

The two types of transplantable methyl cholanthrene induced fibrosarcoma in rats was used to find out the possible relationship between the fibrinolytic, procoagulant activities and the metastasizing capacity of the tumours. The highly metastatic tumour seems to possess high fibrinolytic activity as compared to the low metastatic one. Interestingly enough, it was found that the procoagulant and fibrinolytic activities in the highly metastasising tumour bear inverse relationship with each other in relation to time of tumour growth. The procoagulant activity of the tumour bypasses factor VII and acts at factor X level. The plasminogen activator present in the tumour tissue has been characterized by sephadex G-200 column chromatography and PAGE.

Animals↗
PubMed1989

Enhanced growth of tumour cells in healing colonic anastomoses and laparotomy wounds.

In the past, it has been noted that experimental tumour cells inoculated into the peritoneal cavity or into the lumen of the bowel will grow at a recently formed colonic anastomosis. However, it has previously been unclear whether the healing process enhances tumour growth or whether the presence of a suture line merely allows the tumour cells to gain access to the tissues. In the present study, using the hooded Lister rat, we have confirmed these findings by showing that growth of the syngeneic MC28 sarcoma and OES5 breast carcinoma occurs preferentially at colonic anastomoses and laparotomy wounds after intraperitoneal injection, and at colonic anastomoses after intraluminal injection. In previous studies using the MC28 sarcoma and the OES5 breast carcinoma injected by the intracardiac route (so that tumour cells reach normal and healing tissues in approximately equal numbers) we have shown that tumour growth is enhanced in healing wounds but not in the surrounding normal tissues when cells reach a healing colonic anastomosis or laparotomy wound within 2 h of its formation. Furthermore, by studying the distribution of radiolabelled tumour cells after intracardiac injection, we have calculated that the probability of a tumour cell leading to a deposit in a healing anastomosis or laparotomy wound is increased 1,000 fold compared to normal tissue. No previous studies have combined the data for intracardiac, intraluminal and intraperitoneal injection of tumour cells using the same animal model. We conclude that the same phenomenon of tumour growth enhancement in colonic anastomoses and laparotomy wounds reported after intracardiac injection of tumour cells may well be enhancing tumour growth after intraperitoneal and intraluminal injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Anastomosis, Surgical↗
PubMed1988

Preferential growth of blood-borne cancer cells at sites of trauma--a growth promoting role of macrophages.

Carcinoma and sarcoma after gaining access to the venous circulation are completely trapped in the organs of first encounter, i.e. the lung and vertebrae for cancers draining into the vena cava, or liver for tumours with portal drainage. Systemic dissemination of carcinomas and sarcomas derives from cancer growing in the lung and can be mimicked in experimental rats by injecting cancer cells intra-arterially via the left ventricle. These emboli are arrested in different tissues in proportion to the cardiac output they receive, but the probability of a cancer embolus introduced in this way causing a lesion varies by several orders of magnitude for different tissues. Traumatising tissues by procedures such as surgical incision of the abdomen, gut anastomosis, placing a stitch in the kidney or chemical or surgical partial hepatectomy, greatly enhances the likelihood that a cancer cell trapped in the tissues develops into a metastasis. While several factors contribute to making a wound a more favourable "soil" for metastasis a major mechanism is the release of growth factors from macrophages that have infiltrated the wound.

Animals↗
PubMed1988

Preferential growth of bloodborne cancer cells in colonic anastomoses.

Intracardiac injection, in hooded Lister rats, of syngeneic MC28 sarcoma cells never induced tumour growth in normal bowel. Tumour growth occurred at the site of a colonic anastomosis if surgery preceded tumour injection but not if it followed tumour injection, even by as little as 1 h. Maximum enhancement of tumour growth occurred when the healing process had progressed between 2 and 8 days, with a peak at 5 to 7 days. The enhancing effect was largely over by the time the healing had progressed 14 days. The syngeneic OES5 breast carcinoma also grew at colonic anastomoses when surgery preceded tumour injection by 5 days, but not in normal colon. The MC28 sarcoma also grew at ileal anastomoses but not in the normal ileum after intracardiac injection. By injecting radiolabelled sarcoma cells, an estimate of the probability of a single bloodborne tumour cell lodging at a colonic anastomosis and leading to a tumour deposit was calculated to be of the order of 1:43 whereas the probability of the cell lodging in normal colon and causing a deposit is less than 1:4 x 10(4).

Anastomosis, Surgical↗
PubMed1988

Mechanisms of organ selective tumour growth by bloodborne cancer cells.

The sites of tumour development for 6 rat tumours injected into syngeneic rats via different vascular routes was determined. Xenografts of human tumours were also injected intra-arterially (i.a.) into immunosuppressed rats. Following intravenous (i.v.) and intraportal (i.ptl.) injection of cells tumour colonies localized in lung and liver respectively due to tumour cell arrest. Arterially injected radiolabelled cells disseminated and arrested in a similar distribution to cardiac output and did not 'home' to any organs. Following arterial injection of unlabelled tumour cells colonies grew in many organs. While the pattern of growth for a particular tumour varied with the cell dose, the 'arterial patterns' for all of the tumours studied followed a similar pattern. Some organs (eg adrenals, ovaries and periodontal ligament) were consistently preferred, others (eg skin and skeletal muscle) only supported tumour growth following the delivery of large numbers of cells, while in some tissues (eg spleen and intestines) tumour never grew. Viable tumour cells could be demonstrated by bioassay in many organs for up to 24h after i.a. injection. However tumour growth only occurred in certain organs and the pattern of this growth was not related to the number of tumour cells arrested or their rate of autolysis. This site preference could be expressed quantitatively as the probability of an arrested cell developing into a tumour and was considered a 'soil effect'. Site preference was not directly related to organ vascularity. Organ colonisation was promoted by steroid treatment but the mechanism was unclear and was not secondary to T-cell immunosuppression or prostaglandin synthesis suppression. The adrenal glands were preferred sites of tumour growth but pharmacological manipulation of adrenal function did not alter tumour growth to this organ. Sites of injury and healing were preferred sites of tumour colonisation and this could not be accounted for by increased delivery of tumour cells to these regions. The possibility that the macrophage component of the inflammatory response promoted tumour growth was suggested from studies in which the interval between trauma and inoculation of tumour cells was varied as well as by promotion of intraperitoneal (i.p.) tumour growth by a macrophage infiltrate.

Animals↗
PubMed1988

Chronic hyperdynamic sepsis in the rat: I. Characterization of the animal model.

We describe a new rat model of chronic hyperdynamic sepsis. After control values for weight gain, and food and water intake of each animal were obtained over a 5-day period, male Sprague-Dawley rats weighing 370-425 g were anesthetized, catheterized to allow chronic cardiac-output measurements, and a sterile subcutaneous cavity was formed over the flank area. The animals were allowed a 3-4 day postoperative recovery period. Body weight, food and water intake, and cardiac output were measured daily. Frequent blood samples were withdrawn for bacterial cultures and white cell counts (WBC). On the third and, in some cases, the fourth postoperative day, the subcutaneous cavity was inoculated with 10(9) colony-forming units of Escherichia coli and Bacteroides fragilis. The resulting sepsis was characterized by loss of body weight in spite of normal food and water intake, increased cardiac output, increased WBC, intermittent bacteremia, decreased muscle mass, and decreased cross-sectional area of skeletal muscle myofibrils. Two levels of septic response emerged--moderate and severe. Based on the above-mentioned measurements, it was possible to categorize all long-term septic animals into these two groups. Both groups exhibited cardiac-output, body-weight, and WBC data significantly different from sham controls. Repeated inoculations of the subcutaneous abscess initiated on the third postoperative day resulted in moderate sepsis with no long-term mortality, severe sepsis with 23% mortality over a 3-week period, or a 100% mortality within 4 days, depending on the virulence of the E. coli organisms used. The new model is ideally suited for pathophysiologic studies of sustained, hyperdynamic sepsis.

Animals↗
PubMed1987

Analogs of leukotriene B4: effects of modification of the hydroxyl groups on leukocyte aggregation and binding to leukocyte leukotriene B4 receptors.

The syntheses and agonist and binding activities of 5(S)-hydroxy- 6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (12-deoxy LTB4), 5(S), 12(S)-dihydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (12-epi LTB4), 12(R)-hydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5-deoxy LTB4), 5(R), 12(S)-dihydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5-epi LTB4), 6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5, 12-deoxy LTB4) are described. These leukotriene B4 analogs were all able to aggregate rat leukocytes and compete with [3H]-leukotriene B4 for binding to rat and human leukocyte leukotriene B4 receptors with varying efficacy. The analog in which the 12-hydroxyl group was removed was severely reduced both in agonist action (aggregation) and binding. The epimeric 12-hydroxyl analog demonstrated better agonist and binding properties than the analog without a hydroxyl at this position. In contrast, in the case of the 5-hydroxyl the epimeric hydroxyl analog had greatly reduced agonist and binding activities while the 5-deoxy analog demonstrated potency only several fold less than leukotriene B4 itself. The dideoxy leukotriene B4 analog was more than a thousand fold less active than leukotriene B4 as an agonist and in binding to the leukotriene B4 receptor. These results show that binding to the leukocyte leukotriene B4 receptor requires a hydroxyl group at the 12 position in either stereochemical orientation but that the presence of a hydroxyl at the 5 position is less important. However, the epimeric C5 leukotriene B4 analog clearly interacts unfavourably with the binding site of the leukotriene B4 receptor.

Animals↗