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P Alexander

Publications and source records attributed to P Alexander.

At least 55 records · Page 3Linked to original sources
PubMed1994

Contribution of host-derived growth factors to in vivo growth of a transplantable murine mammary carcinoma.

The contribution of host-derived growth factors to tumour growth in vivo was studied using the transplantable murine mammary carcinoma, MT1, grown in syngeneic mice. Promotion of growth of the mammary carcinoma by a factor(s) from the host was evident in experiments in which the carcinoma cells were inoculated intraperitoneally. In this environment, tumours develop as multiple solid nodules, each probably arising from an individual cell or a small cluster of cells. Tumour growth was found to occur in the peritoneal cavity following inoculation of 10(3) cells, but an inoculum of as few as ten cells grew if a leucocyte-rich exudate had first been induced. To determine which host-derived growth factors might contribute to growth of MT1, extracts of the tumour were first examined for growth factor activity. Fractionation of tumour extracts by either ion-exchange chromatography or gel filtration revealed several peaks of mitogenic activity, but none of this could be attributed to epidermal growth factor (EGF). Accordingly, an anti-EGF antibody was tested as a putative inhibitor of tumour growth as any effect of this antibody could be ascribed to removal of EGF derived from the host. The antibody was found to have potent anti-tumour activity when tested against MT1 tumours that had been inoculated into the peritoneal cavity. In contrast, the antibody had little effect on growth of the discrete tumour mass which formed when MT1 was transplanted subcutaneously. The results suggest that host-derived EGF contributes to establishment of microcolonies of MT1 carcinoma within the peritoneal cavity. This may be directly, by providing growth factors to supplement those produced by the tumour until it reaches a certain critical mass to sustain autocrine growth, or indirectly, by affecting the production of other growth-stimulatory factors or cytokines.

Animals↗
PubMed1994

A double-blind, placebo-controlled, multicenter study with alprazolam and extended-release alprazolam in the treatment of panic disorder.

This is a double-blind, placebo-controlled, flexible-dose, multicenter, 6-week study comparing regular alprazolam (compressed tablet, CT), given four times per day, and extended release alprazolam (XR), given once in the morning. The aim of the XR preparation is to offer less frequent dosing and to reduce interdose anxiety. Of the intent-to-treat group of 209 patients, 184 completed 3 weeks of medication and were evaluated according to protocol. There was a completer rate for the 6 weeks of 94% (CT), 97% (XR), and 87% (placebo). On global measures, Hamilton Rating Scale for Anxiety, phobia rating, and work disability measures, both active treatment groups were equally effective and significantly more efficacious than the placebo cell on endpoint MANOVA analysis. On analysis of the panic factor with endpoint data, both active treatment groups were equally effective throughout the 6-week trial and significantly more efficacious than the placebo group. Drowsiness occurred more frequently with CT alprazolam (86% of patients) than with the XR preparation (79%) or placebo (49%).

Adult↗
PubMed1993

An engineered disulfide cross-link accelerates the refolding rate of calcium-free subtilisin by 850-fold.

The mature form of subtilisin is an unusual example of a monomeric protein with a high kinetic barrier to folding and unfolding. Using site-directed mutagenesis of subtilisin BPN', we are attempting to determine the physical and energetic nature of the kinetic barrier. The high-affinity calcium-binding site A has been shown to create a large enthalpic barrier to unfolding. Removing the calcium-binding site A from subtilisin by deleting amino acids 75-83 greatly accelerates both unfolding and refolding reactions. Here a disulfide cross-link is introduced between residues 22 and 87 in delta 75-83 subtilisin. This was done to probe the conformational entropy of the transition state for folding. The 1.8-A X-ray structure of this mutant and the effects of the cross-link on the kinetics of unfolding and refolding are reported. Consistent with an expected loss of entropy of the unfolded protein due to the cross-link, the disulfide accelerates folding relative to the uncross-linked form. The magnitude of the acceleration of folding rate (700-850-fold at 25 degrees C) indicates that residues 22 and 87 are ordered in the transition state such that the disulfide does not affect its total entropy. Although early organization of structure around amino acids 22 and 87 greatly accelerates folding, we do not know whether the early folding of this region is a highly populated folding pathway in the absence of the cross-link. The slow step in the delta 75-83 subtilisin folding reaction may be forming initial structures capable of propagating the folding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1993

Catalysis of a protein folding reaction: thermodynamic and kinetic analysis of subtilisin BPN' interactions with its propeptide fragment.

The in vivo folding of subtilisin is dependent on a 77 amino acid propeptide, which is eventually cleaved from the N-terminus of subtilisin to create the 275 amino acid mature form of the enzyme (Ikemura et al., 1987). We have cloned, expressed, and purified large quantities of the 77 amino acid subtilisin propeptide. This has enabled us to characterize its participation in the subtilisin folding reaction by spectroscopic and microcalorimetric methods. Unfolded subtilisin, when returned to native conditions, is kinetically isolated from its native state. Folding of subtilisin with the native calcium site-A is extremely slow even in the presence of a high concentration of isolated propeptide. The folding of a calcium-free mutant subtilisin, however, is readily catalyzed by the isolated propeptide. The propeptide-subtilisin folding reaction can be described as the following equilibrium: P(u) + S(u)<==>P-S<==>Pf-Sf<==>P(u) + Sf, where S(u) and P(u) are subtilisin and propeptide, respectively, which are largely unstructured at the start of the reaction; P-S is a collision complex of unfolded subtilisin and propeptide; Pf-Sf is the complex of folded subtilisin and propeptide; and Sf is folded subtilisin. The rate-limiting step in the folding reaction of calcium-free mutant subtilisin is formation of the initial collision complex, P-S. The rate at which P(u) and S(u) form a productive collision complex is approximately 500 M-1 s-1. The collision complex appears to be an early folding unit which, once formed, results in rapid isomerization to the fully folded complex. The rate constant for isomerization of the collision complex to the folded complex is > or = 0.5 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Molteno tube implantation for neovascular glaucoma. Long-term results and factors influencing the outcome.

BACKGROUND: The Molteno implant has been shown to be useful in the treatment of neovascular glaucoma. However, a wide range of success rates has been reported. This is related to the use of differing criteria for success, varying periods of follow-up, and difficulty in quantifying the preoperative condition of the eye. METHODS: The authors studied the long-term results of the Molteno single-plate implant in 60 eyes with neovascular glaucoma using Kaplan-Meier life-table analysis. Age, visual acuity, underlying retinal diseases, and preoperative retinal ablation treatment were evaluated to establish factors influencing the surgical outcome. The criteria for success included a postoperative intraocular pressure (IOP) of less than or equal to 21 mmHg and maintenance of vision. RESULTS: The success rate was 62.1% at 1 year, 52.9% at 2 years, 43.1% at 3 years, 30.8% at 4 years, and 10.3% at 5 years. The main causes for failure were loss of light perception in 48% of eyes (29/60), progression to phthisis bulbi in 18% (11/60), and encapsulation of the filtering bleb in 10% (6/60). The long-term surgical outcome was significantly better in patients older than 55 years of age (P = 0.048) and in those with a preoperative visual acuity equal to or better than 6/60 (P = 0.019). Eyes with neovascular glaucoma secondary to diabetic retinopathy had a better prognosis than those with a central retinal vein occlusion (P = 0.003). CONCLUSION: Although the IOP can be significantly reduced after Molteno implantation, this study suggests that in severely compromised eyes with neovascular glaucoma the main advantage of Molteno implantation is pain relief and avoidance of enucleation.

Adolescent↗
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↗