ABSTRACT
The human interferon alpha2b (hu-IFNalpha2b) gene was cloned in Escherichia coli JM109(DE3) and the recombinant protein was expressed as cytoplasmic inclusion bodies (IB). The present work discusses the recovery of hu-IFNalpha2b IB from the E. coli cells. An optimized protocol is proposed based on the sequential evaluation of recovery steps and parameters: (i) cell disruption, (ii) IB recovery and separation from cell debris, (iii) IB washing, and (iv) IB solubilization. Parameters such as hu-IFNalpha2b purity and recovery yield were measured after each step. The optimized recovery protocol yielded 60% of hu-IFNalpha2b with a purity of up to 80%. The protein was renatured at high concentration after recovery and it was found to display biological activity.
Subject(s)
Escherichia coli/genetics , Inclusion Bodies/chemistry , Inclusion Bodies/genetics , Interferon-alpha/chemistry , Interferon-alpha/genetics , Animals , Chlorocebus aethiops , Cloning, Molecular , Cytoplasm/genetics , Cytoplasm/metabolism , Escherichia coli/cytology , Escherichia coli/metabolism , Humans , Inclusion Bodies/metabolism , Interferon alpha-2 , Interferon-alpha/isolation & purification , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Solubility , Urea/chemistry , Vero CellsABSTRACT
The yield of human alpha 2b interferon in Escherichia coli was optimized by replacement of low-usage arginine codons located in the mRNA 5' end. The differences observed among the various gene variants suggest that codon usage, Shine-Dalgarno-like sequences, and mRNA secondary structure contribute to the performance of E. coli translation machinery.
Subject(s)
Codon , Escherichia coli/metabolism , Interferon-alpha/biosynthesis , Protein Biosynthesis , Amino Acid Sequence , Arginine , Base Sequence , Escherichia coli/genetics , Humans , Interferon alpha-2 , Interferon-alpha/genetics , Molecular Sequence Data , RNA, Messenger/genetics , Recombinant ProteinsABSTRACT
The morphological aspects of the cervix uteri of twelve albino rats, six in estrus and six in diestrus were studied. Micrometry of the epithelium thickness was performed and it was observed that the epithelium is significantly thicker during the estrus phase of the cycle than in diestrus. The major causes for this are discussed and the findings are correlated with the estrogen and progesterone levels present in each of the mentioned phases.
Subject(s)
Cervix Uteri/anatomy & histology , Diestrus , Estrus , Animals , Epithelium/anatomy & histology , Female , Histological Techniques , Rats , Rats, WistarABSTRACT
Seventy-eight women with breast disease were studied aiming to evaluate the importance of the fine needle aspiration biopsy for its diagnosis and forwarding treatment. Our data showed a high diagnostic accuracy with a low level of false negative results and no false positive. The macroscopic appearance of samples is an important aspect. Clear material prevailed in benign pathology (90%) and the presence of blood was usual in malignant tumors (78.3%).
Subject(s)
Breast Diseases/pathology , Biopsy, Needle/instrumentation , Biopsy, Needle/methods , Biopsy, Needle/statistics & numerical data , Breast/pathology , Breast Diseases/epidemiology , Chi-Square Distribution , Cytodiagnosis/methods , Cytodiagnosis/statistics & numerical data , Diagnosis, Differential , False Negative Reactions , False Positive Reactions , Female , Humans , Sensitivity and SpecificityABSTRACT
A simple operation, anterior abdominal wall colpopexy using autogenous strips of rectus fascia, to repair posthysterectomy prolapse of the vaginal vault, is described. It is mainly indicated for patients with complete vaginal eversion, in whom preservation of a physiologically useful vagina is highly desirable. Normal vaginal caliber and depth were secured and no recurrence has arisen in a series of nine patients operated upon for complete prolapse of the vaginal vault after hysterectomy, seven of whom have been followed up for six months to 12 years.
