Individualized Antisense Oligonucleotide Therapies: How to Approach the Challenge of Manufacturing These Oligos from a Chemistry, Manufacturing, and Control-Regulatory Standpoint

2021 ◽  
Author(s):  
Marc M. Lemaitre
Keyword(s):  
Antisense Oligonucleotide ◽  
And Control

scholarly journals An Antisense Oligonucleotide to 1-cys Peroxiredoxin Causes Lipid Peroxidation and Apoptosis in Lung Epithelial Cells

2002 ◽  
Vol 277 (51) ◽  
pp. 49927-49934 ◽  
Author(s):  
Jhang Ho Pak ◽  
Yefim Manevich ◽  
Han Suk Kim ◽  
Sheldon I. Feinstein ◽  
Aron B. Fisher
Keyword(s):  
Lipid Peroxidation ◽  
Antisense Oligonucleotide ◽  
Plasma Membranes ◽  
Apoptotic Cell ◽  
Lung Epithelial Cells ◽  
Epithelial Cell Line ◽  
Positive Staining ◽  
Complementary Sequence ◽  
Lung Epithelial ◽  
And Control

1-cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin superfamily, reduces phospholipid hydroperoxides as well as organic peroxides and H2O2. To determine the physiological function(s) of 1-cysPrx, we have used an antisense strategy to suppress endogenous 1-cysPrx in L2 cells, a rat lung epithelial cell line. A 25-base antisense morpholino oligonucleotide was designed to bind a complementary sequence overlapping the translational start site (−18 to +7) in the rat 1-cysPrx mRNA, blocking protein synthesis. Treatment with an antisense oligonucleotide for 48 h resulted in approximately 60% suppression of the 1-cysPrx protein content as measured by immunoblot analysis and an approximately 44% decrease of glutathione peroxidase activity as compared with random oligonucleotide treated and control (vehicle only) cells. Accumulation of phosphatidylcholine hydroperoxide in plasma membranes was demonstrated by high pressure liquid chromatography assay for conjugated dienes (260 pmol/106cells for antisenseversus70 pmol/106cells for random oligonucleotide and control cells) and by fluorescence of diphenyl-1-pyrenylphosphine, a probe for lipid peroxidation. The percentage of cells showing positive staining for annexin V and propidium iodide after antisense treatment was 40% at 28 h and 80% at 48 h. TdT-mediated dUTP nick end labeling assay at 48 h indicated DNA fragmentation in antisense-treated cells that was blocked by prior infection with adenovirus encoding 1-cysPrx or by pretreatment with a vitamin E analogue. The results indicate that 1-cysPrx can function in the intact cell as an antioxidant enzyme to reduce the accumulation of phospholipid hydroperoxides and prevent apoptotic cell death.

scholarly journals Participation of prolactin receptors and phosphatidylinositol 3-kinase and MAP kinase pathways in the increase in pancreatic islet mass and sensitivity to glucose during pregnancy

2004 ◽  
Vol 183 (3) ◽  
pp. 469-476 ◽  
Author(s):  
Maria E C Amaral ◽  
Daniel A Cunha ◽  
Gabriel F Anhê ◽  
Mirian Ueno ◽  
Everardo M Carneiro ◽  
...  
Keyword(s):  
Map Kinase ◽  
Tyrosine Phosphorylation ◽  
Antisense Oligonucleotide ◽  
Janus Kinase ◽  
Serine Phosphorylation ◽  
Phosphatidylinositol 3 Kinase ◽  
Pregnant Rats ◽  
Islet Mass ◽  
And Control ◽  
Phosphatidylinositol 3

Prolactin (PRL) exerts its biological effects mainly by activating the Janus kinase/signal transducer and activator of transcription 5 (JAK/STAT5) signaling pathway. We have recently demonstrated that PRL also stimulates the insulin receptor substrates/phosphatidylinositol 3-kinase (IRSs/PI3K) and SH2-plekstrin homology domain (SHC)/ERK pathways in islets of neonatal rats. In the present study, we investigated the involvement of the PI3K and MAP kinase (MAPK) cascades in islet development and growth in pregnant rats. The protein expression of AKT1, p70S6K and SHC was higher in islets from pregnant compared with control rats. Higher basal levels of tyrosine phosphorylation were found in classic transducers of insulin cell signaling (IRS1, IRS2 and SHC). Increased levels of threonine/tyrosine phosphorylation of ERK1/2 and serine phosphorylation of AKT and p70S6K were also detected. To assess the participation of PRL in these phenomena, pregnant and control rats were treated with an antisense oligonucleotide to reduce the expression of the PRL receptor (PRLR). Phosphorylation of AKT was reduced in islets from pregnant and control rats, whereas p70S6K protein levels were reduced only in islets from treated pregnant rats. Finally, glucose-induced insulin secretion was reduced in islets from pregnant but not from control rats treated with the PRLR antisense oligonucleotide. In conclusion, downstream proteins of the PI3K (AKT and p70S6K) and MAPK (SHC and ERK1/2) cascades are regulated by PRL signaling in islets from pregnant rats. These findings indicate that these pathways participate in the increase in islet mass and the sensitivity to glucose during pregnancy.

Control of Metal Alloy Oxidation with Electric Fields

1973 ◽  
Vol 31 ◽  
pp. 164-165
Author(s):  
R. R. Dils ◽  
P. S. Follansbee
Keyword(s):  
Electric Fields ◽  
Oxidation Process ◽  
Base Alloy ◽  
Oxide Surface ◽  
Platinum Electrodes ◽  
Insulating Layer ◽  
Iron Base Alloy ◽  
Alloy Oxidation ◽  
Aluminum Oxide Layer ◽  
And Control

Electric fields have been applied across oxides growing on a high temperature alloy and control of the oxidation of the material has been demonstrated. At present, three-fold increases in the oxidation rate have been measured in accelerating fields and the oxidation process has been completely stopped in a retarding field.The experiments have been conducted with an iron-base alloy, Pe 25Cr 5A1 0.1Y, although, in principle, any alloy capable of forming an adherent aluminum oxide layer during oxidation can be used. A specimen is polished and oxidized to produce a thin, uniform insulating layer on one surface. Three platinum electrodes are sputtered on the oxide surface and the specimen is reoxidized.

Evidence for a Blood Ganglion Barrier in the Superior Cervical Ganglion of the Rat

1982 ◽  
Vol 40 ◽  
pp. 204-204
Author(s):  
D. M. DePace
Keyword(s):  
Blood Vessels ◽  
Superior Cervical Ganglion ◽  
Peripheral Nerves ◽  
Time Schedule ◽  
Transmission Electron ◽  
Cervical Ganglion ◽  
The Central Nervous System ◽  
Cervical Ganglia ◽  
Capillary Circulation ◽  
And Control

The majority of blood vessels in the superior cervical ganglion possess a continuous endothelium with tight junctions. These same features have been associated with the blood brain barrier of the central nervous system and peripheral nerves. These vessels may perform a barrier function between the capillary circulation and the superior cervical ganglion. The permeability of the blood vessels in the superior cervical ganglion of the rat was tested by intravenous injection of horseradish peroxidase (HRP). Three experimental groups of four animals each were given intravenous HRP (Sigma Type II) in a dosage of.08 to.15 mg/gm body weight in.5 ml of.85% saline. The animals were sacrificed at five, ten or 15 minutes following administration of the tracer. Superior cervical ganglia were quickly removed and fixed by immersion in 2.5% glutaraldehyde in Sorenson's.1M phosphate buffer, pH 7.4. Three control animals received,5ml of saline without HRP. These were sacrificed on the same time schedule. Tissues from experimental and control animals were reacted for peroxidase activity and then processed for routine transmission electron microscopy.

Effects of renovascular hypertension on rat adrenal zona glomerulosa

1978 ◽  
Vol 36 (2) ◽  
pp. 582-583
Author(s):  
G. Mazzocchi ◽  
P. Rebuffat ◽  
C. Robba ◽  
P. Vassanelli ◽  
G. G. Nussdorfer
Keyword(s):  
Renovascular Hypertension ◽  
Left Kidney ◽  
Renin Angiotensin System ◽  
Plasma Renin ◽  
Zona Glomerulosa ◽  
Ultrastructural Changes ◽  
Albino Rats ◽  
Left Renal Artery ◽  
Angiotensin System ◽  
And Control

It is well known that the rat adrenal zona glomerulosa steroidogenic activity is controlled by the renin-angiotensin system. The ultrastructural changes in the rat zona glomerulosa cells induced by renovascular hypertension were described previously, but as far as we are aware no correlated biochemical and morphometric investigations were performed.Twenty adult male albino rats were divided into 2 experimental groups. One group was subjected to restriction of blood flow to the left kidney by the application of a silver clip about the left renal artery. The other group was sham-operated and served as a control. Renovascular hypertension developed in about 10 days: sistolic blood pressure averaged 165 ± 6. 4 mmHg, whereas it was about 110 ± 3. 8 mmHg in the control animals. The hypertensive and control rats were sacrificed 20 days after the operation. The blood was collected and plasma renin activity was determined by radioimmunological methods. The aldosterone concentration was radioimmunologically assayed both in the plasma and in the homogenate of the left capsular adrenal gland.

X-Ray Replication of Submicrometer Linewidth Patterns

1977 ◽  
Vol 35 ◽  
pp. 136-137
Author(s):  
Henry I. Smith ◽  
D.C. Flanders
Keyword(s):  
Electron Beam Lithography ◽  
Cross Sectional ◽  
X Ray ◽  
Electron Backscattering ◽  
Spatial Frequencies ◽  
Cross Sectional Profile ◽  
Carbon Foils ◽  
Sectional Profile ◽  
And Control ◽  
Soft Radiation

Scanning electron beam lithography has been used for a number of years to write submicrometer linewidth patterns in radiation sensitive films (resist films) on substrates. On semi-infinite substrates, electron backscattering severely limits the exposure latitude and control of cross-sectional profile for patterns having fundamental spatial frequencies below about 4000 Å(l),Recently, STEM'S have been used to write patterns with linewidths below 100 Å. To avoid the detrimental effects of electron backscattering however, the substrates had to be carbon foils about 100 Å thick (2,3). X-ray lithography using the very soft radiation in the range 10 - 50 Å avoids the problem of backscattering and thus permits one to replicate on semi-infinite substrates patterns with linewidths of the order of 1000 Å and less, and in addition provides means for controlling cross-sectional profiles. X-radiation in the range 4-10 Å on the other hand is appropriate for replicating patterns in the linewidth range above about 3000 Å, and thus is most appropriate for microelectronic applications (4 - 6).

Ultrastructural changes in sciatic nerve tissue: Effects of passive maternal smoking

1983 ◽  
Vol 41 ◽  
pp. 650-651
Author(s):  
Amankwah K.S. ◽  
A.D. Weberg ◽  
R.C. Kaufmann
Keyword(s):  
Sciatic Nerve ◽  
Maternal Smoking ◽  
Ultrastructural Level ◽  
Uranyl Acetate ◽  
Nerve Tissue ◽  
Ultrastructural Changes ◽  
Cacodylate Buffer ◽  
And Control ◽  
Spontaneous Delivery

Previous research has revealed that passive (involuntary inhalation) tobacco smoking during gestation can have adverse effects upon the developing fetus. These prior investigations did not concentrate on changes in fetal morphology. This study was undertaken to delineate fetal neural abnormalities at the ultrastructural level in mice pups exposed in utero to passive maternal smoking.Pregnant study animals, housed in a special chamber, were subjected to cigarette smoke daily from conception until delivery. Blood tests for determination of carbon monoxide levels were run at 15-18 days gestation. Sciatic nerve tissue from experimental and control animals were obtained following spontaneous delivery and fixed in 2.5% gluteraldehyde in 0.1M cacodylate buffer pH 7.3. The samples were post-fixed in osmium ferrocyanide (1:1 mixture of 1.5% aqueous OSO4 and 2.5% K4 Fe(CN)6). Following dehydration, the tissues were infiltrated with and embedded in Spurr. Sections were stained with uranyl acetate and lead citrate.

A Superconducting Microscope

1971 ◽  
Vol 29 ◽  
pp. 10-11 ◽  
Author(s):  
R. E. Worsham ◽  
J. E. Mann ◽  
E. G. Richardson
Keyword(s):  
Liquid Helium ◽  
Focal Length ◽  
Vacuum System ◽  
Objective Lens ◽  
Electrical Instability ◽  
Radiation Shields ◽  
And Control ◽  
Thermal Oscillations ◽  
Flux Pump

This superconducting microscope, Figure 1, was first operated in May, 1970. The column, which started life as a Siemens Elmiskop I, was modified by removing the objective and intermediate lenses, the specimen chamber, and the complete vacuum system. The large cryostat contains the objective lens and stage. They are attached to the bottom of the 7-liter helium vessel and are surrounded by two vapor-cooled radiation shields.In the initial operational period 5-mm and 2-mm focal length objective lens pole pieces were used giving magnification up to 45000X. Without a stigmator and precision ground pole pieces, a resolution of about 50-100Å was achieved. The boil-off rate of the liquid helium was reduced to 0.2-0.3ℓ/hour after elimination of thermal oscillations in the cryostat. The calculated boil-off was 0.2ℓ/hour. No effect caused by mechanical or electrical instability was found. Both 4.2°K and 1.7-1.9°K operation were routine. Flux pump excitation and control of the lens were quite smooth, simple, and, apparently highly stable. Alignment of the objective lens proved quite awkward, however, with the long-thin epoxy glass posts used for supporting the lens.

Morphogenesis of a New Human Herpes-Type Virus Isolate (Sasha Virus)

1973 ◽  
Vol 31 ◽  
pp. 592-593
Author(s):  
R. F. Zeigel ◽  
W. Munyon
Keyword(s):  
Virus Isolate ◽  
Calf Serum ◽  
Uranyl Acetate ◽  
Human Herpes ◽  
Human Embryonic Lung ◽  
Human Herpes Virus ◽  
Intracytoplasmic Inclusions ◽  
Hel Cells ◽  
And Control

In continuing studies on the role of viruses in biochemical transformation, Dr. Munyon has succeeded in isolating a highly infectious human herpes virus. Fluids of buccal pustular lesions from Sasha Munyon (10 mo. old) uiere introduced into monolayer sheets of human embryonic lung (HEL) cell cultures propagated in Eagles’ medium containing 5% calf serum. After 18 hours the cells exhibited a dramatic C.P.E. (intranuclear vacuoles, peripheral patching of chromatin, intracytoplasmic inclusions). Control HEL cells failed to reflect similar changes. Infected and control HEL cells were scraped from plastic flasks at 18 hrs. of incubation and centrifuged at 1200 × g for 15 min. Resultant cell packs uiere fixed in Dalton's chrome osmium, and post-fixed in aqueous uranyl acetate. Figure 1 illustrates typical hexagonal herpes-type nucleocapsids within the intranuclear virogenic regions. The nucleocapsids are approximately 100 nm in diameter. Nuclear membrane “translocation” (budding) uias observed.

Residual Gas Reaction in the Electron Microscope: II The Design of a Gas-Control Chamber for Quantitative Observations

1969 ◽  
Vol 27 ◽  
pp. 82-83
Author(s):  
Chester J. Calbick ◽  
Richard E. Hartman
Keyword(s):  
Electron Beam ◽  
Electron Microscope ◽  
Local Environment ◽  
Chamber Pressure ◽  
Objective Lens ◽  
Ion Pump ◽  
Quantitative Studies ◽  
Precise Knowledge ◽  
Differential Pumping ◽  
And Control

Quantitative studies of the phenomenon associated with reactions induced by the electron beam between specimens and gases present in the electron microscope require precise knowledge and control of the local environment experienced by the portion of the specimen in the electron beam. Because of outgassing phenomena, the environment at the irradiated portion of the specimen is very different from that in any place where gas pressures and compositions can be measured. We have found that differential pumping of the specimen chamber by a 4" Orb-Ion pump, following roughing by a zeolite sorption pump, can produce a specimen-chamber pressure 100- to 1000-fold less than that in the region below the objective lens.

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