Biomedical Engineering Reference
In-Depth Information
Functional studies were performed in
Xenopus
oocytes injected with water, hPepT1
cRNA, the 208 hPepT1 splice variant cRNA, and hPepT1 cRNA combined with the
hPepT1 splice variant cRNA. Interestingly, results demonstrated a lack of transport
activity for Gly-Sar in hPepT1-RF-injected oocytes; however, cotransfection of the
hPepT1 splice variant with hPepT1 affected the pH sensitivity of peptide uptake in
the double transfect, suggesting a pH-regulatory function for the splice variant and
leading to its hPepT1-RF name.
Studies by Sadee and co-workers reported multiple splice variants in the hPHT1
gene.
63
In several regions of the hPHT1 sequence deduced, multiple expressed se-
quence tag (EST) overlap was observed, suggesting possible sequence variations in
the human population. Expression analysis in several tissues and by EST alignments
suggests the presence of possible hPHT1 splice variants, as indicated by an insert
or gap in two regions of the
hPHT1
coding sequence. Indeed, our laboratory iden-
tified a previously unreported hPHT1 splice variant from Caco-2 cells utilizing the
primers designed for the cloning of the hPHT1 full-length sequence.
65
This PHT1
cDNA contained a gap of 58 bp between bases 842 and 900 from the start codon.
This gap induced a shift in the coding frame, resulting in a predicted protein sequence
of 295 amino acids.
65
Interestingly, this hPHT1 splice variant comprised the first
six conserved hPHT1 TMDs, similar to the hPepT1-RF,
161
raising potential ques-
tions concerning modularity in the hPHT1 gene coding information and its functional
relevancy.
13
Recently, Pinsonneault et al. reported haplotype analysis of the PepT2 gene to
determine the effect of variants on PepT2 expression and function.
162
Among these
variants, PepT2*1 and *2 were shown to be significantly present in 247 human ge-
nomic DNA samples from different ethnic groups. A haplotype is the combination of
several sequence variants on a single chromosome at a specific locus and may more
accurately reflect genetic diversity in the human population. Wild-type hPepT2 and
its variants were transfected into CHO cells and their function was investigated after
24 hours. Results indicated significantly different
K
m
values for Gly-Sar uptake for
each variant. Furthermore, varying levels of hPepT2*1 and *2 mRNA were observed
in nine heterozygous kidney tissue samples, suggesting the presence of
cis
-acting
polymorphisms affecting transcription or mRNA processing.
6.5. PHARMACEUTICAL DRUG SCREENING
Permeation of peptide-based compounds across cellular barriers can occur by several
routes (Figure 6.1). One needs to be mindful that each of these routes of permeation
can apply to different peptide-based compounds; however, the physicochemical prop-
erties of the compound and the physiological significance of each route will determine
the relative importance in controlling the compound's net absorption. In screening
compound permeation across cellular barriers, delineation of the key rate-determining
steps for controlling absorption needs to be identified. Methods to delineate the func-
tional relevancy of each route are now being enhanced further by focusing the kinetics
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