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M. Kuczer et al. / Bioorganic Chemistry 66 (2016) 12–20
of 0.07 mg/ml. Each spectrum represents the average of four scans.
The data are presented as molar ellipticity [h].
the reduction of the virus titer in the presence of compounds inves-
tigated as compared with the control. TCID50/ml (median tissue
culture infective dose) was calculated. TCID50/ml denotes the
amount of a pathogenic agent that will produce pathological
changes in 50% of cell virus-inoculated cultures. Each analysis
was performed at least in triplicate.
2.4. Cells
Vero cell line was used in this study. Cells were grown and
maintained at 37 °C in Eagle’s medium 1959 (Biomed, Lublin) sup-
plemented with 10% FBS from Gibco and 1% of antibiotic antimy-
cotic solution (100ꢀ): penicillin, streptomycin, amphotericin B
(Sigma-Aldrich) at 37 °C.
2.9. Apoptosis assay
Bioassays with insect hemocytes were performed as described
previously [9,23]. The T. molitor beetles were anaesthetized with
CO2, washed in distilled water, and disinfected with 70% ethanol.
2.5. Virus
The peptide solution was injected to the beetles by hand (2 ll, in
The viral strain used in this study was the standard (reference)
strain of Human Herpes Virus 1 (HHV-1) named McIntyre strain
(HHV-1MC). The herpesvirus stock was propagated in Vero cells.
After the cytopathic effect was evident, the cells were frozen-
thawed three times. The cell debris was removed by centrifugation.
The supernatant was aliquoted, titrated, and kept at ꢁ70 °C. In the
antiviral assay, the medium was supplemented with 2% FBS and
the above mentioned antibiotics.
a dose of 10 nM of peptide per insect) through the ventral mem-
brane between the second and the third abdominal segments
toward the head with a Hamilton syringe (Hamilton Co., Bonaduz,
Switzerland). The control insects were injected with the same vol-
ume of physiological saline. All solutions were sterilized by filtra-
tion through the 0.22 lm pore filter membrane (Millipore) and
all injections were performed in sterile conditions. The peptide
concentration (1 mM stock solution) was assessed before filtration
and next a working solution, which was injected to the beetles, was
prepared.
2.6. Insects
Before hemolymph collection, the beetles were anaesthetized
again with CO2, washed in distilled water, and disinfected with
70% ethanol. Hemocytes from control and insects injected with
peptides were collected 1 h after injection. Upon cutting off a tar-
Studies were carried out on adults beetles, T. molitor L. which
were maintained in laboratory cultures (Department of Animal
´
Physiology and Development, Poznan University). The T. molitor
was reared as described previously [9]. As the mealworm parents’
age is important for the developmental features of their offspring
[19–21], all insects in our experiments are derived from younger
than 1 month parents.
sus from a foreleg, hemolymph samples (5
‘‘end to end” microcapillaries (Drummond). They were diluted
with 20 l of ice-cold physiological saline containing anticoagulant
ll), were collected with
l
buffer (4.5 mM citric acid and 9 mM sodium citrate) at a 5:1 v/v
ratio. Hemolymph from control and peptide-injected insects was
2.7. In vitro cytotoxicity assay
placed on alcohol-cleaned cover glasses coated with 7
ll 0.01%
poly- -lysine (Sigma P4707). After allowing hemocytes to settle
L
Cytotoxic activity of peptides was assessed by a light inverted
microscopy Olympus CK2 (Olympus Corp., Germany) and quanti-
fied by the MTT (Sigma-Aldrich) colorimetric assay in vitro using
a Vero cell line. The absorbance was read spectrophotometrically
at a wavelength of 405 nm on a reader (Reader 230, Organon Tech-
nica Turnhout, Belgium). Cells were inoculated in a 96-microwell
plate. After incubation for 24 h, the peptides were added to the cul-
ture medium in doses ranging from 50 to 500 lg/ml and incubated
for further 24 or 48 h. The control was prepared without any sam-
ple. All experiments were performed in triplicate.
(15 min, at room temperature), the remaining fluid was removed
and the cells were washed twice with physiological saline. The pre-
pared hemocytes were used for the microscopic analysis of activa-
tion of caspases.
The presence of active caspases was investigated by using a sul-
forhodamine derivative of valylalanylaspartic acid fluoromethyl
ketone, a potent inhibitor of caspase activity (SR-VAD-FMK) (in
accordance with the manufacturer’s instructions of the sulforho-
damine multi-caspase activity kit, AK-115, BIOMOL, PA). Hemo-
cytes were incubated in a reaction medium (1/3x SR-VAD-FMK)
for 1 h at room temperature in the dark, rinsed three times with
a wash buffer for 5 min at a room temperature, and finally fixed
in 3.7% paraformaldehyde for 10 min. After washing again in phys-
iological saline, the hemocytes were stained with DAPI (40,6-diami
dino-2-phenylindole). Incubation in the dark lasted for 5 min.
Thereafter, the hemocytes were washed once with distilled water
and mounted using a mounting medium. The prepared hemocytes
were studied with a Nikon Eclipse TE 2000-U fluorescence micro-
scope with filters set for rhodamine (excitation 543 nm and emis-
sion 560 nm) and the intensity of fluorescence of the apoptosing
cells was measured by using an NIS-Element AR 3.1 programme.
The changes in fluorescence intensity were used to quantify the
activity of caspases. Data are shown as mean SD.
2.8. Antiviral activity assay
Antiviral activity was assessed in vitro by phenotypic assays
using a Vero cell line. Cell culture in flat-bottom microwell plates
(2 ꢀ 104 cells/0.2 ml) was infected with an HHV-1MC strain (0.01
TCID50/cell) for 60 min at 37 °C. After the virus absorption, the
inoculum was aspired and fresh culture medium containing a pep-
tide was added.
After a virus infection the cells were incubated for 2 days at
37 °C with various concentrations of the respective compounds,
ranging from 50 to 500 lg/ml diluted in an assay medium.
The antiviral activity of the peptides was determined using a
cytopathic effect (CPE). The inhibition of the viral CPE was assessed
by a light inverted microscopy. The yield reduction assay (YRA),
which measures the inhibition of infectious virus in the presence
of the investigated peptides was applied. Virus titers were deter-
mined according to the Reed-Muench formula [22] and expressed
in TCID50/ml. The Reed-Muench statistical method was used to
determine the 50% end point (EC50) which was the lowest concen-
tration of the tested drugs that reduce the viral infections of the
control to 50%. The antiviral effect was estimated according to
3. Results and discussion
In this work, the structure-activity relation of the insect peptide
alloferon was studied to understand the structural requirements
for its biological activity.
In the literature, many strategies have been suggested to study
structure-activity relationship of biologically active compounds
Kuczer, Mariola
