Creation of a test plasmid for detecting G-C-to-T-A transversions by changing serine to arginine in the active site of beta-lactamase.

Oligonucleotide-directed mutagenesis of the beta-lactamase gene, bla, on pBR322 was used to change the codon for the active-site serine 70, AGC, to CGC, coding for arginine. Escherichia coli cells carrying the mutant plasmid, pGD104, were sensitive to ampicillin, indicating that the arginine-containing enzyme is inactive. We characterized the reversion of the mutant bla gene by a number of mutagens and in different genetic backgrounds and demonstrated that full ampicillin resistance can be restored only by a G-C-to-T-A transversion occurring at the first base of the codon. Thus, reversion of the mutant bla gene is diagnostic for G-C-to-T-A transversions, and bacteria carrying pGD104 can be used as test strains to detect the occurrence of this mutation.     

Metabolically inactive 3T3 cells can substitute for marrow stromal cells to promote the proliferation and development of multipotent haemopoietic stem cells

When highly enriched multipotential spleen colony forming cells (CFU-S) obtained following fluorescence activated cell sorting (FACS-CFU-S) are cultured on marrow stromal cells, they undergo proliferation and development to produce mature haemopoietic cells (Spooncer et al., Nature, 316:62-64, 1985). We now show that FACS-CFU-S behave in a similar way when cultured on monolayers of 3T3 cells, indicating that the 3T3 cells can supply at least part of the environment which is representative of marrow stromal cells and provide, therefore, a system for studying stromal cell: haemopoietic cell interactions. We also demonstrate that IL-3-dependent multipotential stem cell lines (FDCP-Mix), but not a variety of other "committed" IL-3-dependent cell lines, resemble FACS-CFU-S in terms of their ability to proliferate and differentiate when cultured on 3T3 cells in the absence of IL-3. In this system, attachment of the FDCP-Mix to the 3T3 cells is critical for the subsequent maintenance of viability and stimulation of development of the cells. When the FDCP-Mix cells are physically separated from the 3T3 cells, they die and their death cannot be prevented by using 3T3-cell-conditioned medium. The extracellular matrix generated by 3T3 cells is not sufficient for promoting attachment or viability of the FDCP-Mix cells, indicating the importance of integral membrane components. However, attachment and development of FDCP-Mix cells occurs on 3T3 cells that have been lightly fixed with glutaraldehyde indicating that active metabolism is not essential for the effects promoted by the 3T3 cells. We suggest that the ability of FACS-CFU-S and FDCP-Mix cells to respond to 3T3 cells involves specific ligand/receptor interactions.     

Molecular mapping of class II polymorphisms in the human major histocompatibility complex. I. DR beta

We have studied 27 cell lines homozygous by consanguinity for the major histocompatibility complex to establish the restriction fragment length polymorphism (RFLP) patterns seen with six different restriction enzymes (Bam HI, Bg1 II, Eco RI, Hinc II, Hind III, Pvu II) and DR beta chain probes. The probes used were a full-length cDNA DR beta probe and a probe specific for the 3' untranslated region. The RFLP obtained represent the first standard patterns for the individual haplotypes DR1 through 7 and DR9 as defined by genetically homozygous lines. The patterns obtained reflect the DR specificities closely, as well as the DRw52 and DRw53 specificities. These latter specificities are associated with the most prominent patterns of RFLP. Bands are present which are unique for the haplotypes DR1, DR2, DR4, DR7, DRw52, and DRw53, and could be used for typing these haplotypes in heterozygotes. Subtypes can be identified for all of the haplotypes except DR1. These subtypes indicate that there is an extensive amount of polymorphism in the DR subregion that has not been identified serologically.     

Superinduction of T lymphocyte-endothelial cell (EC) binding by treatment of EC with interleukin 1 and protein synthesis inhibitors

We have previously reported that marked enhancement of the in vitro binding of lymphocytes to endothelial cell (EC) monolayers is observed after stimulation of the EC with interleukin 1 (IL 1). To determine whether new protein synthesis was required for this effect of IL 1, EC were incubated with IL 1 in the presence of cycloheximide or puromycin. Three different effects of these protein synthesis inhibitors on T-EC binding were observed. First, preincubation of the EC with both IL 1 and an inhibitor blocked the increase in binding if the inhibitor was present during both the preincubation and the 1 hr duration of the T-EC binding assay, suggesting that new protein synthesis is required for the enhancement of T-EC adhesion by IL 1. Second, preincubation of the EC with low doses of the inhibitors (0.1 to 1 microgram/ml) in the absence of IL 1 consistently increased T-EC binding, even if the inhibitors were present during the T-EC adhesion assay; in addition, the inhibitors additionally increased the stimulatory effect of IL 1 if the EC were washed free of the inhibitor before the assay step. The binding-enhancing effect of low concentrations of cycloheximide could be inhibited by an antibody to the CDw18 complex on the T cell, suggesting an up-regulation of the ligand on the EC involved in CDw18-dependent T cell adhesion. Third, higher concentrations of the inhibitors (3 to 10 micrograms/ml) were toxic for the EC in the presence of IL 1, possibly due to the combined blocking effect of IL 1 and inhibitors on EC protein synthesis.     

Characterization of the phospholipid and fatty acid composition of Sendai virus

The lipid composition of Sendai virus, propagated in chicken eggs, was analyzed by high performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and gas-liquid chromatography (GLC). Phosphatidylcholine was found to be the dominant phospholipid (37.3%) with phosphatidylethanolamine (26.8%) and phosphatidylserine (12.0%) also present in significant amounts. Analysis of the fatty acid methyl esters revealed that the dominant fatty acids in total phospholipid were: C16:0 (17.6%), C18:0 (15.4%), C18:1 (n-9) (22.0%), and C24:0 (6.0%). Cardiolipin, phosphatidylserine, and sphingomyelin contained higher levels of saturated fatty acids relative to phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine.     

Fine needle aspiration of the breast. Importance of the aspirator

All 503 fine needle aspirations (FNAs) of the breast performed over a three-year period at the Medical Center Hospital of Vermont were analyzed. There were 93 aspirates diagnosed as "positive," all of which were from patients eventually shown to have cancer. However, there were 38 patients with primary carcinoma in which the FNA was not diagnosed as positive, for a diagnostic failure rate of 31.4%. In order to determine the possible effect of technique as practiced by an experienced aspirator in diminishing such diagnostic failures, we compared 190 aspirates obtained by a single individual with 193 aspirates obtained by 15 individuals in the same community. For the single experienced aspirator, the technical failure rate was 9.8% whereas in the group with many aspirators it was 45.9%. This finding confirms that, although fine needle aspiration of the breast is considered easy to perform, skill on the part of the aspirator is important for satisfactory results.     

Behavioral responses of red hake,Urophycis chuss,to decreasing concentrations of dissolved oxygen

Synopsis Laboratory experiments were conducted to examine changes in behavior of red hake,Urophycis chuss, under decreasing concentrations of dissolved oxygen (DO). Since the ecological requirements of this species change with age, responses were measured for three different groups: (1) age 0+, = 89 mm total length (TL); (2) age 1+, = 238 mm TL; and (3) age 2–3+, = 397 mm TL. As DO decreased from 8–10 mg l^-1 to < 0.5 mg l^-1, changes were evident in active time, water column activity, range of horizontal movement, food searching, and agonistic behavior. Age 0+ fish were most sensitive, moving up into the water column and swimming continuously as DO levels fell below 4.2 mg l^-1. Age 2–3+ fish were the least responsive, remaining on the substrate and increasing only their range of movement at concentrations below 3 mg l^-1. Responses of age 1 + fish were variable, possibly reflecting a transition stage between the younger and older fish. Common to all groups was the decrease and eventual cessation of food searching.     

Sex determination from the radius in humans

The discriminatory capacity of the radius in sex determination was investigated in a Dutch skeletal collection of recent origin. Midshaft subperiosteal diameter, maximum length and maximum transverse distal width were measured from roentgenograms. The discriminatory capacity of the radius was found to be of the same order as that reported in the literature for the other long bones. Maximum transverse distal width showed the highest consistency (85%) between estimated and documented sex. This method requires only the presence of the distal fragment of the radius.     

Planktivory in Pagothenia borchgrevinki (Pisces: Nototheniidae) in McMurdo sound,Antarctica

Summary Specimens of the Antarctic fish P. borchgrevinki were collected from just beneath the sea-ice in McMurdo Sound over the period 8 November to 6 December, 1985. The stomach contents of 200 fish were analysed and compared with the co-occurrence of the prey species in the plankton. Numerically, the dominant prey was a thecosomatid pteropod Limacina helicina (83%), followed by a hyperiid amphipod Hyperiella dilatata (5%). Other prey species in decreasing order of occurrence were Euphausia crystallorophias, Euchaeta antarctica, other copepods, a decapod crustacean larva, chaetognaths, the amphipods Orchomene plebs and Epimiriella macronyx, and unidentified juvenile fish. The order of frequency of occurrence of these prey in the guts of all the fish was H. dilatata (in 73% of the fish), L. helicina (71%), calanoid copepods (55%), chaetognaths (51%), E. crystallorophias (42%), decapod crustacean larva (32%), O. plebs (27%), juvenile fish (20%), and E. macronyx (14%). In volumetric terms, the dominant diet contributors were O. plebs (38%), L. helicina (17%) and chaetognaths (15%). All prey except O. plebs and E. macronyx were taken in concurrent plankton samples. Although planktivory of Pagothenia borchgrevinki is confirmed, it is not certain whether feeding is confined to the immediate sub-ice environment.     

Propeptide of human protein C is necessary for gamma-carboxylation

Protein C is one of a family of vitamin K dependent proteins, including blood coagulation factors and bone proteins, that contains gamma-carboxyglutamic acid. Sequence analysis of the cDNAs for these proteins has revealed the presence of a prepro leader sequence that contains a pre sequence or hydrophobic signal sequence and a propeptide containing a number of highly conserved amino acids. The pre region is removed from the growing polypeptide chain by signal peptidase, while the pro region is subsequently removed from the protein prior to secretion. In the present study, deletion mutants have been constructed in the propeptide region of the cDNA for human protein C, and the cDNAs were then expressed in mammalian cell culture. These deletions included the removal of 4, 9, 12, 15, 16, or 17 amino acids comprising the carboxyl end of the leader sequence of 42 amino acids. The mutant proteins were then examined by Western blotting, barium citrate adsorption and precipitation, amino acid sequence analysis, and biological activity and compared with the native protein present in normal plasma. These experiments have shown that protein C is readily synthesized in mammalian cell cultures, processed, and secreted as a two-chain molecule with biological activity. Furthermore, the pre portion or signal sequence in human protein C is 18 amino acids in length, and the pro portion of the leader sequence is 24 amino acids in length. Also, during biosynthesis and secretion, the amino-terminal region of the propeptide (residues from about -12 through -17) is important for gamma-carboxylation of protein C, while the present data and those of others indicate that the carboxyl-terminal portion of the propeptide (residues -1 through -4) is important for the removal of the pro leader sequence by proteolytic processing.