Rhinovirus 3C protease catalyzes efficient cleavage of a fluorescein-labeled peptide affording a rapid and robust assay.

The 3C protease encoded by human rhinovirus type 2 catalyzes with equal efficiency cleavage of a peptide substrate with or without a fluorescein label attached to the amino acid at the P7' position. Substrates Ac-MEALFQGPLQYKDL-NH2 and MEALFQGPLQYKE(fluorescein)L are hydrolyzed with values of Vmax/KM of 970 M-1 s-1 and 1100 M-1 s-1, respectively. With the labeled substrate, HPLC achieves separation of substrate and product in 2.5 min. Separation in as little as 12 s is feasible. Fluorescein was derivatized so that it could be incorporated into peptides using automated solid-phase peptide synthesis.     

Phosphorylation of insulin-like growth factor I receptor by insulin receptor tyrosine kinase in intact cultured skeletal muscle cells

The interaction between insulin and insulin-like growth factor I (IGF I) receptors was examined by determining the ability of each receptor type to phosphorylate tyrosine residues on the other receptor in intact L6 skeletal muscle cells. This was made possible through a sequential immunoprecipitation method with two different antibodies that effectively separated the phosphorylated insulin and IGF I receptors. After incubation of intact L6 cells with various concentrations of insulin or IGF I in the presence of [32P]orthophosphate, insulin receptors were precipitated with one of two human polyclonal anti-insulin receptor antibodies (B2 or B9). Phosphorylated IGF I receptors remained in solution and were subsequently precipitated by anti-phosphotyrosine antibodies. The identities of the insulin and IGF I receptor beta-subunits in the two immunoprecipitates were confirmed by binding affinity, by phosphopeptide mapping after trypsin digestion, and by the distinct patterns of expression of the two receptors during differentiation. Stimulated phosphorylation of the beta-subunit of the insulin receptor correlated with occupancy of the beta-subunit of the insulin receptor by either insulin or IGF I as determined by affinity cross-linking. Similarly, stimulation of phosphorylation of the beta-subunit of the IGF I receptor by IGF I correlated with IGF I receptor occupancy. In contrast, insulin stimulated phosphorylation of the beta-subunit of the IGF I receptor at hormone concentrations that were associated with significant occupancy of the insulin receptor but negligible IGF I receptor occupancy. These findings indicate that the IGF I receptor can be a substrate for the hormone-activated insulin receptor tyrosine kinase activity in intact L6 skeletal muscle cells.     

Alternate pathways of DNA replication in Escherichia coli

We have described the pcbA1 mutation which enables E. coli cells to replicate DNA in the absence of a functional dnaE gene product if DNA polymerase I (the polA gene product) is present. The pcbA1 mutation phenotypically suppresses multiple dnaEts and dnaEam alleles. The pcbA1/PolI replication pathway differs from normal in sensitivity to certain DNA-damaging agents such as methylmethane sulfonate (MMS) and a lack of damage-directed mutagenesis. We report here cloning of the pcbA1 gene in a multicopy plasmid. The pcbA1 mutation is detected only in cis; therefore, cloning necessitated gene eviction. The pcbA1 gene lies closely- linked to gyrB. We have demonstrated the physical presence of DNA polymerase I in the replicating holoenzyme complex by immunoblotting using dnaEam strains. We conclude that E. coli has two alternate replisome structures: REP-A, in which DNA polymerase I is the functional synthetic subunit; and REP-E, in which the alpha-subunit, product of the dnaE gene, is functional. To investigate further the role of individual DNA polymerases in replication, we have isolated the polB gene on multicopy plasmids.     

Ecological comparison of the pre- and post-impoundment macrophyte flora of the river Niger and Lake Kainji,Nigeria

The floristic composition of the macrophyte vegetation of Lake Kainji was investigated and compared with the vegetation of the river Niger before impoundment.Of 18 hydatophytes, 40 tenagophytes and 57 trichophytes listed for the Niger, 18 tenagophytes, 12 trichophytes and 7 hydatophytes were found growing in the lake 17 years after impoundment. Fourteen new tenagophytes, 3 new hydatophytes and 1 new trichophyte were recorded. The hydatophytes which were expected to colonize the lake are limited in number and range.The failure of the hydatophytes to colonize the lake, as predicted by Cook (1968), is discussed in relation to the inflow-to-volume ratio of the lake.Nomenclature follows Hutchinson & Dalziel (1952–1972). Flora of West Tropical Africa.     

Characterization of monoclonal antibodies to the IGF-I receptor that inhibit IGF-I binding to human cells

A mouse monoclonal antibody directed against the human placental IGF-I receptor is shown to completely inhibit IGF-I binding to both human placental membranes and circulating human mononuclear leukocytes. Insulin binding is unaffected. Studies with 125I-labelled anti-receptor antibody indicate that the antibody reacts with an epitope distinct from the IGF-I binding site. These antibodies will be powerful probes of IGF-I binding and action in human cells.     

Traditional preparation and uses of cassava in Nigeria

Various methods of cassava preparation are practised by different ethnic groups in Nigeria. These methods involve peeling cassava roots, soaking roots in streams, grating cassava, and pressing grated cassava. Other methods include heating sieved, grated cassava, boiling peeled cassava roots, and pounding boiled or dried cassava roots. The traditional, cassava-based products aregari, fufu, akpu, cassava flour, edible starch, and tapioca. Detoxification of fresh cassava roots is partly achieved through cell rupture during cutting and grating, soaking in running or standing water in earthen pots for 3–5 days, heating, drying, and boiling.     

Lack of correlation between differentiation status and response to radiation of three murine squamous cell carcinomas

Summary The gross growth rate, histology, cellular kinetics, andin situ radiobiological response have been measured for three murine, keratinising squamous cell carcinomas that differed in their degree of differentiation. Growth rate was fastest in the least-differentiated tumour, slowest in the best-differentiated. However, the kinetics of the compartment of undifferentiated cells that are likely to be radiotherapeutically important, were the same for the three lines. There was no correlation between degree of differentiation and intrinsic or apparent radiosensitivity as measured by the growth delay assay. The radiobiologically best-oxygenated tumour was that which had the largest stromal component and this was not the best-differentiated tumour.     

Longitudinal roentgencephalometric study of the growth of the New Zealand white rabbit: cumulative and biweekly incremental growth rates for skull and mandible

A longitudinal roentgencephalometric study of the New Zealand white rabbit has documented postnatal skull and mandible growth from 2 to 34 weeks of age. Dorsoventral and lateral radiographs were performed using a specially constructed restrainer that allowed for standardized films and did not require the use of anesthesia. Assessments from 17 male and 12 female rabbits at biweekly intervals documented cumulative growth as well as biweekly incremental growth. Indices reported here include skull length, interzygomatic width, intercondylar width, and mandibular length. Mean skull length at 2 weeks was 54% of that at 34 weeks, and by 16 weeks 91% of adult skull length was achieved. Mean interzygomatic width at 2 weeks was 60% of that at 34 weeks, and by 16 weeks 91% of adult male and 94% of adult female widths were achieved. Mean mandibular length at 2 weeks was 47% of that at 34 weeks, and by age 16 weeks 90% of adult length was achieved. Mean intercondylar width at 2 weeks was 57% of that at 34 weeks, and by 16 weeks 89% of adult male and 93% of adult female widths were achieved. Biweekly increments decreased continually from 2 weeks of age on for all indices.     

Inhibition of in vitro germination and spherulation ofCoccidioides immitis byAllium sativum

An aqueous extract of a dehydrated garlic preparation with uniform consistency inhibited all eight clinical isolates of the dimorphic fungus,Coccidioides immitis. The inhibitory and lethic concentrations were in the range of 3.12–6.25 mg/ml for both the saprophytic (mold) and parasitic (spherule) forms ofC. immitis. At 6.25-mg/ml concentration, the organism lost its viability within 6 h. The conversion of arthroconidia into spherules in a chemically defined liquid medium was prevented by garlic extract diluted to 1:320 (3.12 mg/ml). The data indicate that components of garlic readily inhibited the in vitro germination and spherulation of this medically important dimorphic fungus.     

Repair response of human fibroblasts to bleomycin damage

The ability of human fibroblasts to repair the specific types of DNA damage caused by bleomycin (BLM) was examined in whole-cell experiments. The method utilized for analysis was alkaline sucrose-gradient centrifugation of DNA. The results of these studies show that a repair pathway exists for the damage produced in DNA by bleomycin. DNA from BLM-treated cells shows a decrease in molecular weight, caused by chemical or enzymatic incision at sites of drug action. If the drug is removed, the DNA rapidly returns to high molecular weight, demonstrating reformation of damaged DNA. This repair response to BLM-damage was also confirmed in fibroblasts isolated from patients with putative DNA-repair defects. We observed that the response (to BLM) of cells from patients with Fanconi anemia was altered in that the fall in molecular weight of DNA from treated cells was not as great as that observed in other cell strains after drug treatment.