Peptides uptake

In this work we will focus on the use of the cubic phase as a delivery system for oligopeptides - Desmopressin, Lysine Vasopressin, Somatostatin and the Renin inhibitor H214/03. The amino acid sequences of these peptides are given in Table I. The work focuses on the cubic phase as a subcutaneous or intramuscular depot for extended release of peptide drugs, and as a vehicle for peptide uptake in the Gl-tract. Several examples of how the peptide drugs interact with this lipid-water system will be given in terms of phase behaviour, peptide self-diffusion, in vitro and in vivo release kinetics, and the ability of the cubic phase to protect peptides from enzymatic degradation in vitro. Part of this work has been described elsewhere (4-6). 

Lactic streptococci initiate casein degradation through the action of cell wall-associated and cell membrane-associated proteinases and peptidases. Small peptides are taken into the cell and hydrolyzed to their constituent amino acids by intracellular peptidases (Law and Sharpe 1978). Peptides containing four to seven residues can be transported into the cell by S. cremoris (Law et al. 1976B). S. lactis and S. cremoris have surface-bound peptidases and thus are not totally dependent on peptide uptake for protein use (Law 1979B). Some surface peptidases of S. cremoris are located in the cell membrane, whereas others are located at the cell wall-cell membrane interface (Exterkate 1984). Lactic streptococci have at least six different aminopeptidase activities, and can be divided into three groups based on their aminopeptidase profiles (Kaminogawa et al 1984). 

Table 7-3 Summary of taxon-specific nitrogen (NO3, NO2, NH +, urea, dissolved free amino acids [DFAA], peptides) uptake rates made in natural systems... 

I, dissolved free amino acids [DFAA], peptides) uptake rates... 

LAB characteristically have substantial numbers of peptide uptake and efflux systems, used for nutrition, signaling, regulation, and biological warfare. All LAB secrete proteins via Sec/Oxal systems, but they lack the Sec auxiliary proteins, SecDF. Because of their rapid growth rates, they may use ATP to drive secretion rather than the proton motive force (pmf) (Tsukazaki et al. 2011). Moreover, all LAB seem to have competence-related and septal DNA translocation proteins, although competencies for DNA uptake are not a demonstrated characteristic of these organisms. Finally, all LAB examined have at least one (and sometimes more) peptidoglycan (murein) precursor exporters. 

To illustrate this, imagine that we are comparing the absorption of glucose with a small peptide. If we measure mass transfer at the same infusion rate (i.e., at equal intestinal flow), we will usually find different unstirred layer thicknesses. There are several reasons why this could occur. First, while glucose is controlled by uptake in the intestinal lumen, peptide uptake may be controlled by transport across the intestinal wall. Even if mass transfer of both solutes is controlled by rates in the lumen, the mass transfer coefficient rarely depends linearly on the diffusion coefficient D. A more typical dependence is on the square root of D. If this is so, then the unstirred layer thickness I depends on D, which makes little sense physically. 

To obtain an increased intrinsic capacity to transgress biological membranes, a number of different modifications have been introduced to PNA. These modifications include conjugation of PNA to Hpophilic moieties [51, 97, 98], conjugation of PNA to certain so-caUed ceU-penetrating peptides [49, 55, 56, 66, 99-102] and conjugation to different moieties, which are supposed to be internahzed by specific cellular receptors [48, 103-105]. The work on cellular dehvery of PNA is, like the related work on ex vivo and in vivo effects of PNA, very difficult to summarize conclusively. First of all, the pronounced diversity of the reporter systems employed makes it impossible to directly compare the studies. Secondly, the widespread use of fluorescence studies in spite of the many inherent pitfalls of this technique makes it sometimes difficult to judge even qualitatively whether a presented result actually indicates cellular uptake. We have recently published a comprehensive review on cellular dehvery of PNA [82], with a more detailed assessment of the PNA dehvery hterature. 

Cell permeabilization and uptake of anti-sense peptide-peptide nucleic acid (PNA) into Escherichia coli. J. Biol. Chem. 2002 277 7144-7147. 

Ljungstrom T., Knudsen H., Nielsen P.E. Cellular uptake of adamantyl conjugated peptide nucleic acids. Bioconjug. 

Koppelhus U., Awasthi S.K., Zachar V., Holst H.U., Ebbesen P., Nielsen P. E. Cell-dependent differential cellular uptake of PNA, peptides, and PNA-pep-tide conjugates. Antisense Nucleic Acid Drug Dev. 2002 12 51-63. 

Basu S., Wickstrom E. Synthesis and characterization of a peptide nucleic acid conjugated to a D-peptide analog of insu-lin-like growth factor 1 for increased cellular uptake. Bioconjug. Chem. 1997 8 481-488. 

Oehlke and coworkers have described the cellular uptake properties of a simple a-hehcal amphipathic model peptide sequence (Lys-Leu-Ala-Leu-Lys-Leu-Ala-Leu-Lys-Ala-Leu-Lys-Ala-Ala-Leu-Lys-Leu-Ala) in the context of a drug delivery vehicle [72]. On the basis of the data presented, it was proposed that non-endocytosis mechanism(s) were involved in the uptake into mammalian cells. The similarity between our b2 aPNA-sequence to that of this amphipathic model peptide makes it tempting to suggest that a similar uptake mechanism is involved in the cellular uptake of aPNAs. Further experimentation is necessary to test this hypothesis. 

The cellular/molecular mechanism of action for these cyclic peptide toxins is now an area of active research in several laboratories. These peptides cause striking ultrastructural changes in isolated hepatocytes (95) including a decrease in the polymerization of actin. This effect on the cells cytoskeletal system continues to be investigated and recent work indirectly supports the idea that these toxins interact with the cells cytoskeletal system (86,96). Why there is a specificity of these toxins for liver cells is not clear although it has been suggested that the bile uptake system may be at least partly responsible for penetration of the toxin into the cell (92). 

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