Добірка наукової літератури з теми "Bromobimane"

The spermatozoa of most marsupials lack nuclear stabilising disulfide-bonded protamines found in eutherian mammals. However, disulfide stabilisation has been observed in the acrosome of macropodid (Macropus eugenii) and phalangerid (Trichosurus vulpecula) marsupials. As a result this organelle, which is normally fragile in eutherian mammals, is robust and able to withstand physical and chemical challenge in these marsupials. The present study examined acrosomal characteristics of the spermatozoa of three dasyurid marsupials; the fat-tailed dunnart (Sminthopsis crassicaudata), eastern quoll (Dasyurus viverrinus) and northern quoll (Dasyurus hallucatus). In all species examined Bryan’s staining demonstrated that significant acrosomal loss occurred following physical challenge with osmotic stress, cryopreservation without cryoprotectant and exposure to detergent (Triton-X). Bromobimane staining indicated that the acrosomes of dasyurids lacked stabilising disulfide bonds. As reported for the wallaby and possum, calcium ionophore (A23187) did not induce the acrosome reaction-like exocytosis in dasyurid spermatozoa but treatment with diacylglycerol (DiC8) caused significant acrosome loss at concentrations similar to those effective for other marsupials. The present study found that the spermatozoa of dasyurids are more sensitive to physical challenge than the previously-studied marsupials and we suggest that this is due to the absence of acrosomal stabilising disulfide bonds.

Abstract We describe a 6-min HPLC method to measure the total concentrations of the most important thiols in plasma and urine–cysteine, homocysteine, cysteinylglycine, and glutathione–as well as the concentrations in plasma and urine, respectively, of cysteamine and 2-mercaptopropionylglycine, two compounds used to treat disorders of cysteine metabolism. Precolumn derivatization with bromobimane and reversed-phase HPLC were performed automatically by a sample processor. Throughput was up to 100 samples in 24 h. The within-run CV ranged from 0.9% to 3.4% and the between-run CV ranged from 1.5% to 6.1%. Analytical recovery was 97–107%, with little difference between plasma and urine samples. The detection limit was ∼50 nmol/L for all the analytes studied. Thiol concentrations were determined in the plasma of 206 healthy donors and in the urine of 318 healthy donors distributed for age and sex. Mean values of plasma cysteine and homocysteine were significantly lower in infants (ages, <1 y) compared with other age groups (P <0.005). In adults, mean plasma homocysteine values were higher in males than in females (9.2 vs 6.7 μmol/L, P <0.0001) and in the 6- to 10-year-old group (P <0.05). Mean values for glutathione and cysteinylglycine were not sex- and age-dependent. In urine, both cysteine and homocysteine showed a wide range of variation.

Type II pneumocytes, which synthesize, store, and secrete pulmonary surfactant, require exogenous fatty acids, in particular palmitic acid, for maximum surfactant synthesis. The uptake of palmitate by type II pneumocytes is thought to be protein mediated, but the protein involved has not been characterized. Here we show by RT-PCR and Northern blot analysis that rat type II pneumocytes express the mRNA for fatty acid translocase (FAT/CD36), a membrane-associated protein that is known to facilitate the uptake of fatty acids into adipocytes. The deduced amino acid sequence from rat type II pneumocytes reveals 98% identity to the FAT/CD36 sequence obtained from rat adipocytes. The uptake of palmitate by type II pneumocytes follows Michaelis-Menten kinetics (Michaelis-Menten constant = 11.9 ± 1.8 nM; maximum velocity = 62.7 ± 5.8 pmol ⋅ min−1 ⋅ 5 × 105pneumocytes−1) and decreases reversibly under conditions of ATP depletion to 35% of control uptake. Incubation of cells at 0°C inhibited the uptake of palmitate almost completely, whereas depletion of potassium was without effect. Preincubation of the cells with bromobimane or phloretin decreases the uptake of palmitate significantly as does preincubation with sulfo- N-succinimidyl oleate, the specific inhibitor of FAT/CD36 (C. M. Harmon, P. Luce, A. H. Beth, and N. A. Abumrad. J. Membr. Biol. 121: 261–268, 1991). From these data, we conclude that FAT/CD36 is expressed in type II pneumocytes and mediates the uptake of palmitate in a saturable and energy-dependent manner. The data suggest that the uptake process is independent of the formation of coated pits and endocytotic vesicles.

Aberrant protein-protein interactions often result in disease, and as such, effective protein-protein interaction inhibitors are needed to mitigate the disease state. These interaction interfaces often involve secondary structural motifs, for example, an α-helix or β-sheet. Small molecule drugs are not well suited to inhibit protein-protein interactions however constrained peptides, have shown to have great therapeutic potential. Short peptides display little secondary structure in aqueous solution and as such, peptide sequences derived from a protein-protein interaction interface for use as a protein-protein interaction inhibitor, must be constrained into the native secondary structure. This can be achieved by installing a linker between the side-chains of two appropriately spaced amino-acids in the sequence. Many different linker chemistries have been designed and implemented with good biological results. However, these constrained peptide therapeutics are still restricted by traditional small molecule drug hurdles including cell permeability, protease degradation and the ability to visualise and track a molecule intracellularly. Linkers such as the all-hydrocarbon metathesis linker have shown great promise in reducing protease degradation and increasing cell permeability, however a fluorescent tag is still necessary to visualise a drug candidate. Here, a bimane linker is proposed as a new peptide linker to help overcome these limitations. Dibromobimane is reacted with thiol-containing amino-acid side chains to introduce a new fluorescent constraint in a series of model peptides. The reaction conditions with dibromobimane are optimised in solution to reveal that a buffered system is required for the cyclisation to occur efficiently. Optimal reaction conditions, determined by monitoring the increase of the fluorescent product, were 0.5 mg/ml peptide in 10 mM PBS with one equivalent of dibromobimane. The reaction was shown to be facile and versatile; in this thesis an array of peptides with varied sequence length, constraint length and amino-acid composition were cyclised under the same conditions, all reaching reaction completion in under 30 minutes. Additionally, these same conditions were applied successfully to react monobromobimane with series of short peptides. Cyclisation on reaction with dibromobimane, was also demonstrated on-resin with similar efficiency. The fluorescent properties of the resultant peptides were then explored to reveal that pH does not affect the observed fluorescence however a longer peptide length resulted in greater fluorescence intensity. Furthermore, acyclic mono-bimane-functionalised peptides displayed lower fluorescence intensity than the bimane-cyclised counterparts. The fluorescence of the bimane cyclised peptide could be detected as low as 10 nM on a plate reader, which is expected to further improve on a more sensitive instrument. The secondary structure of a series of tri- and penta-peptides were investigated through CD and NMR techniques. It was deduced that the bimane linker can induce β-strand like structure in an i-i+2 constrained peptide; in contrast an i-i+4 constrained pentapeptide with homocysteine in the 1 and 5 positions results in a 3₁₀ helical like structure. β-alanine containing analogues of these peptides were also synthesised and showed minimal structure.This work outlines the synthesis of macrocyclic peptides containing a peptide constraint, in the form of a fluorescent bimane, both in solution and on-resin to produce cyclised peptides. The fluorescent properties of the resultant peptides have been shown to be biologically compatible with great fluorescence sensitivity. Furthermore, different secondary structure can be introduced by simply alterations of the constraint length from i-i+2 to i-i+4. This work provides a foundation on which to design new fluorescent bimane-cyclised peptide-based protein-protein interaction inhibitors.
Thesis (M.Phil.) -- University of Adelaide, School of Physical Sciences, 2017.