Efficient extraction of RNA from mammalian tissue

RNA extraction from mammalian tissue has been compared using the different deproteinizing agents: a) guanidine-HCl, b) guanidinium-thiocyanate, c) buffer-saturated phenol, or d) buffer-saturated phenol followed by a proteinase K digestion of the aqueous phase. Both solid tissues (first, second, and third trimester fetal bovine pancreas), and human white blood cell populations were studied. Degradation, as seen in citric acid-urea agarose gels, and the ability to serve as templates for cell-free protein synthesis were used as criteria to assess the efficiency of the different methods. We conclude that employing buffer-saturated phenol with proteinase K digestion is a superior method for consistent extraction of relatively undegraded RNA in quantitative amounts from mammalian tissue.     

Comparative analysis of multiple protein-sequence alignment methods [published erratum appears in Mol Biol Evol 1994 Sep;11(5):811]

We have analyzed a total of 12 different global and local multiple protein-sequence alignment methods. The purpose of this study is to evaluate each method's ability to correctly identify the ordered series of motifs found among all members of a given protein family. Four phylogenetically distributed sets of sequences from the hemoglobin, kinase, aspartic acid protease, and ribonuclease H protein families were used to test the methods. The performance of all 12 methods was affected by (1) the number of sequences in the test sets, (2) the degree of similarity among the sequences, and (3) the number of indels required to produce a multiple alignment. Global methods generally performed better than local methods in the detection of motif patterns.      

Mutation of the C/EBP binding sites in the Rous sarcoma virus long terminal repeat and gag enhancers.

Several C/EBP binding sites within the Rous sarcoma virus (RSV) long terminal repeat (LTR) and gag enhancers were mutated, and the effect of these mutations on viral gene expression was assessed. Minimal site-specific mutations in each of three adjacent C/EBP binding sites in the LTR reduced steady-state viral RNA levels. Double mutation of the two 5' proximal LTR binding sites resulted in production of 30% of wild-type levels of virus. DNase I footprinting analysis of mutant DNAs indicated that the mutations blocked C/EBP binding at the affected sites. Additional C/EBP binding sites were identified upstream of the 3' LTR and within the 5' end of the LTRs. Point mutations in the RSV gag intragenic enhancer region, which blocked binding of C/EBP at two of three adjacent C/EBP sites, also reduced virus production significantly. Nuclear extracts prepared from both chicken embryo fibroblasts (CEFs) and chicken muscle contained proteins binding to the same RSV DNA sites as did C/EBP, and mutations that prevented C/EBP binding also blocked binding of these chicken proteins. It appears that CEFs and chicken muscle contain distinct proteins binding to these RSV DNA sites; the CEF binding protein was heat stable, as is C/EBP, while the chicken muscle protein was heat sensitive.     

Fetal circulation during epidural analgesia for caesarean section.

Fetal blood flow was examined during epidural analgesia in six women with uncomplicated pregnancies undergoing elective caesarean section. A non-invasive, ultrasonic technique was used to measure blood flow in the fetal descending aorta and intra-abdominal part of the umbilical vein before induction of analgesia with etidocaine and bupivacaine and 15 and 30 minutes afterwards. No appreciable change in fetal blood flow was observed.     

In vitro stimulation of alkaline phosphatase activity in immature embryonic chick pelvic cartilage by adenosine

Cyclic AMP content in embryonic chick pelvic cartilage increases significantly as the embryo ages from 8 to 10 d. This in ovo elevation in cyclic AMP content precedes maximal cartilage alkaline phosphatase activity by some 24 h. We studied whether this temporal relationship may be causally related, using an in vitro organ culture. Incubation of pelvic cartilage from 9- and 10-d embryos in medium containing monobutyryl cyclic AMP (BtcAMP) resulted in significant increases in alkaline phosphatase activity (220 and 66 percent, respectively) as compared to that of cartilages incubated in medium alone. This stimulation was both concentration- and time-dependent with maximal response at 0.5 mM BtcAMP and 4-h incubation, respectively. Similar incubations of cartilage in medium containing 1-methyl-3-isobutyl xanthine (MIX), 0.25 mM, also resulted in increased alkaline phosphatase activity (114 percent). However, pelvic cartilage from 11-d embryos incubated in medium containing BtcAMP or MIX showed no increase in alkaline phosphatase activity. We postulated that developmental age was the factor responsible for this difference in response and that immature cartilage (that with little or no alkaline phosphatase activity) would respond to BtcAMP whereas mature cartilage (that with significant alkaline phosphatase activity) would not. This was tested by incubating end sections of 11-d cartilage, which have little alkaline phosphatase activity, and center sections, which have significantly alkaline phosphatase activity, with both BtcAMP and MIX. Alkaline phosphatase activity in end sections (immature cartilage) was stimulated by BtcAMP and MIX, whereas it was not stimulated in the center sections. Actinomycin D and cycloheximide inhibited BtcAMP and MIX stimulation of alkaline phosphatase activity. Thus, the in vitro data suggest that cyclic AMP is a mediator for the stimulation of alkaline phosphatase activity in embryonic cartilage.     

The isolation and identification of multiple forms of the neutrophil granule peptides from human leukemic cells

HP-1 is a 30-residue cysteine- and arginine-rich peptide of the human neutrophil primary granule and is the most abundant human representative of the family of peptides variously called defensins and corticostatins. Peptides belonging to this family have many biological activities including the non-oxidative destruction of ingested microorganisms, the inhibition of adrenocorticotropin-stimulated synthesis of glucocorticoids, monocyte chemotaxis, the non-cytolytic inhibition of [3H]thymidine incorporation in HL-60 promyelocyte-like cells and the stimulation of nifedipine-sensitive calcium channels. Using a combination of reversed-phase and size-exclusion high performance liquid chromatography and an HP-1 radio-immunoassay, three immunoreactive peptides were detected and isolated from the promyelocyte-like cell line, HL-60, and from leukocytes of patients with chronic myelogenous and chronic lymphocytic leukemias. One of these peptides was HP-1 itself. A second was identified by gas-phase Edman microsequencing as an amino-terminally extended fragment of the HP-1 precursor which we call HP1-56. The third is likely to arise from enzymatic cleavage of the precursor at a dibasic site. Of the leukemic cells the greatest amount of HP1-56 relative to HP-1 was found in cells from a patient in myeloblastic crisis but overall the richest source of HP1-56 relative to HP-1 was found to be in fetal lung tissue. HP1-56 is difficult to detect in normal peripheral neutrophils and its presence in cells that are actively biosynthesizing primary granule components such as HL-60 may make it useful for studying the biosynthesis of granule polypeptides, their ontogeny, and possibly as a marker protein for leukemic diseases.     

Defective lysis of streptomycin-resistant escherichia coli cells infected with bacteriophage f2

A lysis defect was found to account for the failure of a streptomycin-resistant strain of Escherichia coli to form plaques when infected with the male-specific bacteriophage f2. The lysis defect was associated with the mutation to streptomycin resistance. Large amounts of apparently normal bacteriophage accumulated in these cells. Cell-free extracts from both the parental and mutant strains synthesized a potential lysis protein in considerable amounts in response to formaldehyde-treated f2 RNA but not in response to untreated RNA. As predicted from the nucleotide sequence of the analogous MS2 phage, the protein synthesized in vitro had the expected molecular weight and lacked glycine. The cistron for the lysis protein overlapped portions of the coat and replicase cistrons and was translated in the +1 reading frame. Initiation at the lysis protein cistron may be favored by translation errors that expose the normally masked initiation site, and streptomycin-resistant ribosomes, known to have more faithful translation properties, may be unable to efficiently synthesize the lysis protein.     

Fetal and neonatal breathing movements in man after betamethasone.

Nineteen women in the last trimester of pregnancy were treated with betamethasone 12 mg daily for three consecutive days. The fetal breathing movements were monitored by an ultrasonic method before and after the treatment and the patterns of movements were compared to those in controls. In seven treated cases the recording of breathing was performed also in the neonatal period. Betamethasone in the given dose, which causes pronounced alterations in the fetal corticosteroid balance, does not influence the pattern of breathing movements in the fetus and newborn. Although corticosteroids are known to affect several functions of CNS, the maturation of mechanisms regulating breathing movements in the perinatal period is apparently not accelerated by betamethasone.