Maternal diabetes is linked with additional likelihood of growth disturbances and congenital malformations. The malformations rate in the offspring of diabetic mothers is 2-3 fold greater when compared with infants of nondiabetic mothers. In this report you’ll find we have researched the function of the protein kinase C (PKC) pathway and the apoptotic machinery in embryopathy. We looked into the involvement of PKC isoforms in the embryopathy of diabetic rat pregnancy. Embryos of diabetic rats revealed transformed activity and protein distribution of several PKC isoforms in contrast to embryos of normal rats. Making use of whole embryo culture we discovered elevated activity of PKC-delta and PKC-zeta after 24h of culture and increased rate of malformations and growth retardation in embryos cultured in high glucose concentration when compared with embryos cultured in low glucose concentration. Addition of a-cyano-4-cinnamic acid and N-acetylcysteine to the culture medium normalized malformations and growth retardations whereas specific PKC-inhibitors abolished malformations and partially renewed the growth retardations……
Diabetes and Pregnancy
Contents
Introduction
Diabetes mellitus
Background
Diabetic pregnancy
Protein kinase C
PKC-delta
PKC-zeta
Apoptosis
p53
Bcl-2
Caspases
Oxidative stress
Treatment
Vitamin E
Folic acid
N-acetylcysteine
Į-cyano-4-cinnamic acid
Embryonic development
Streptozotocin
Animal model
Aims
Material & Methods
Animals
Treatment (Paper III)
Preparation of total RNA (Paper I & II)
Preparation of cDNA (Paper I & II)
Analysis of mRNA levels (Paper I & II)
Estimation of PKC activity markers (Paper I and IV)
Fractionation of embryonic cells
Immunoblot analysis
Measurement of protein
Western blot analysis (Paper II & III)
Immunostaining of embryos (Paper I & II)
Whole embryo culture (Paper II and IV)
Culture of embryonic cells (Paper II)
Analysis of Nuclear factor-ț B activation (Paper III)
Extraction of nuclear proteins
Electromobility shift assay
Detection of cell death and apoptosis (Paper II)
Activated Caspase 3 detection in living cells
Propidium iodide flow cytometry
Vital staining with propidium iodide and Hoechst 33342
Statistical considerations
Results and discussion…………..
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