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.     

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.     

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.     

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.     

Improvement in separation characteristics of protein precipitates by acoustic conditioning

The effect of acoustic conditioning on the particle size distribution of isoelectric and calcium-ion-precipitated soya protein has been examined in low-residence-time chambers. In a previous study a beat frequency of 5 Hz obtained using a dual-source system of opposing vibrators was determined as giving optimal improvement in particle-settling characteristics for isoelectric soya protein precipitate. In this study the effect of amplitude of vibration, a measure of acoustic power input, and residence time of acoustic conditioning has been examined.Acoustic power input changed the flow pattern in the conditioning chamber from laminar streamline flow to a well-mixed, turbulent pattern. Such a mixing effect promoted the rapid aggregation of fine particles, a process that was modeled on the basis of orthokinetically controlled collisions. The rate of removal of fine particles due to acoustic conditioning was shown to be proportional to a mixing effect that was releated to the acoustic power dissipated per unit volume.The consequences of fine-particle aggregation on the centrifugal recovery of the precipitate are discussed.     

Formation of acetylcarnitine in muscle of horse during high intensity exercise

To study the changes in carnitine in muscle with spring exercise, two Thoroughbred horses performed two treadmill exercise tests. Biopsies of the middle gluteal were taken before, after exercise and after 12 min recovery. Resting mean muscle total carnitine content was 29.5 mmol.kg-1 dry muscle (d.m.). Approximately 88% was free carnitine, 7% acetylcarnitine and acylcarnitine was estimated at 5%. Exercise did not affect total carnitine, but resulted in a marked fall in free carnitine and almost equivalent rise in acetylcarnitine. The results are consistent with a role for carnitine in the regulation of the acetyl-CoA/CoA ratio during sprint exercise in the Thoroughbred horse by buffering excess production of acetyl units.     

Immunocytochemical markers revealing retinal and pineal but not hypothalamic photoreceptor systems in the Japanese quail

Summary The retinal proteins opsin,-transducin, S-antigen and interstitial retinol-binding protein (IRBP) are essential for the processes of vision. By use of immunocyto-chemistry we have employed antibodies directed against these photoreceptor proteins in an attempt to identify the photoreceptor systems (retina, pineal and deep brain) of the Japanese quail. Opsin immunostaining was identified within many outer (basal portion) and inner segments of retinal photoreceptor cells and limited numbers of photoreceptor perikarya. Opsin immunostaining was also demonstrated in limited numbers of pinealocytes with all parts of these cells being immunoreactive. These results differ from previous observations. In contrast to the results obtained with the antibody against opsin, S-antigen and-transducin immunostaining was seen throughout the entire outer segments and many photoreceptor perikarya of the retina. In the pineal organ immunostaining was seen in numerous pinealocytes in all follicles. These results conform to previous findings in birds. In addition, IRBP has been demonstrated for the first time in the avian retina and pineal organ. These findings underline the structural and functional similarities between the retina and pineal organ and provide additional support for a photoreceptive role of the avian pineal. No specific staining was detected in any other region of the brain in the Japanese quail; the hypothalamic photoreceptors of birds remain unidentified.     

Metabolism and excretion of exogenous [3H]-LTC4 in primates

Four novel omega- and beta-oxidation (from the omega end) products of peptide leukotrienes, 20-hydroxy and 20-carboxy-LTE4, 18-carboxy-19, 20-dinor-LTE4 and 16-carboxy-17,18,19,20-tetranor-14,15-dihydro-LTE4 were prepared by total synthesis and used as standards for identification of biliary and urinary metabolites in the cynomolgus monkey. After intravenous administration 14, 15-[3H] leukotriene C4 (10 microCi kg-1) was partially metabolized in and rapidly cleared from the vascular circulation. This resulted, within 24 hours, in significant urinary excretion (14.8 +/- 2.1%, n = 4), consisting largely of material more polar than LTE4 (61% of urinary excretion) as shown by reverse phase HPLC. The polar fraction demonstrated two predominant metabolites which coeluted in several HPLC solvent systems with synthetic 16-carboxytetranordihydro-LTE4 (major component) and 18-carboxydinor-LTE4 (minor component). Characterization of the major polar metabolite as 16-carboxytetranordihydro-LTE4 was substantiated by conversion to its N-acetylated derivative. The absence of the 14, 15 double bond was confirmed by product analysis of oxidative ozonolysis. In a single animal, the bile duct was cannulated, with significant biliary excretion of radioactivity demonstrated over 4 hours (58.6% recovery). The predominant polar biliary metabolites were also identified as the 18-carboxydinor and 16-carboxytetranordihydro derivatives of LTE4 mentioned above. These data suggest that beta-oxidation products generated from the omega-carboxyl end of the 20-carboxy-LTE4 are important products of [3H] LTC4 metabolism in the monkey. Quantitation of these urinary metabolites may be an important index of in vivo leukotriene production.