Synthesis of epidermal growth factor (EGF) receptor in vitro using SP6 RNA polymerase-transcribed template mRNA

The epidermal growth factor (EGF) receptor plays a key role in the control cellular proliferation, and its homology to the avian erythroblastosis virus erb B oncogene implicates its involvement in cellular transformation. The establishment of a correlation between the various structural domains of the EGF receptor and their functional counterparts would greatly advance our understanding of these processes. To this end, we have constructed an expression vector containing the SP6 viral promoter and an adjacent cDNA fragment encoding the full-length EGF receptor. Upon addition of SP6 RNA polymerase, this DNA is capable of generating large amounts of EGF receptor mRNA; this RNA can then be translated in vitro into immunoprecipitable EGF receptor protein. The translational efficiency of this EGF receptor RNA was found to be relatively low: approx. 100-fold lower than globin RNA synthesized using SP6 RNA polymerase. Use of these tools should now permit the synthesis and analysis of mutated EGF receptor protein in an effort to clarify the role of this receptor in growth control.     

Eimeria tenella, E. necatrix, E. acervulina, E. maxima, and E. brunetti: potent anticoccidial activity of an uridine analog, 1-(beta-D-ribofuranosyl)-2(1H)-pyrazinone 4-oxide

The anticoccidial activity of an uridine analog, 1-(beta-D-ribofuranosyl)-2(1H)-pyrazinone 4-oxide (emimycin riboside), against five species of chicken Eimeria was tested individually in battery experiments. With 16 ppm of the compound in feed, marked anticoccidial activity was obtained against Eimeria tenella, E. necatrix, E. acervulina, E. maxima, and E. brunetti. The last named species was more drug-sensitive than the others--dietary levels of at least 8 ppm of the drug exhibited good protection and eliminated practically all clinical signs. The battery tests with delayed and restricted medications showed that emimycin riboside affected the development of parasites in first and second generation schizogony of the life cycle of E. tenella.     

5C6-F4, a novel 100,000-dalton rat lymphocyte activation antigen defined by monoclonal antibody

Con A-activated rat thymocytes were used to immunize mice, and immune spleen cells were fused with NS/1 myeloma cells. One clone, designated 5C6-F4, reacted strongly with Con A-activated rat thymocytes and some LPS-activated rat spleen cells but not with normal thymocytes, spleen cells, or bone marrow cells of rat origin. The 5C6-F4 did not react with Con A-activated thymocytes of mouse origin. Immunoprecipitation of 5C6-F4 antigen from surface-iodinated Con A-activated rat thymocytes or LPS-activated rat spleen cells revealed its m.w. to be approximately 100,000. The kinetic studies of the expression of 5C6-F4 antigen revealed that 5C6-F4 antigen was detectable at 6 hr after Con A stimulation of rat spleen cells, whereas IL 2 receptor (IL 2R) was detectable at 12 hr. The appearance of 5C6-F4 antigen and IL 2R precede the onset of DNA synthesis of Con A-activated spleen cells. Thus, 5C6-F4 antigen is classified as early activation antigen. The 5C6-F4 inhibits the lymphocyte proliferation induced by mitogen and the IL 2-driven rat T cell proliferation. Sequential immunoprecipitation study as well as binding inhibition study indicated that the 5C6-F4 antigen is distinct from IL 2R molecule. The 5C6-F4 antigen appears to be a novel rat lymphocyte activation antigen that exhibits immunoregulatory function and also may serve as a useful marker of T cell activation.     

Mutagenic activation of aflatoxin B1 by several forms of purified cytochrome P-450

Metabolic activation by several forms of purified cytochrome P-450 of aflatoxin B1 to a product(s) mutagenic to Salmonella typhimurium TA100 was examined. Of the 5 forms of cytochrome P-450 purified from liver microsomes of untreated and PCB-treated male rats, a constitutive form purified from untreated male rats, P-450-male, and a high-spin form of cytochrome P-450, P-448-H, from PCB-treated rats were highly active.     

Mouse lymphoma L1210 cells acquire a new cystine transport activity upon adaptation in vitro

Summary Mouse lymphoma L1210 cells maintained in vitro at a high cell density for a certain time period adapted themselves to the in vitro environment and were able to grow indefinitely. From these adapted cells, more than 30 clones were isolated. They all had much higher activity to take up cystine than the original L1210 cells, supporting a previous view that the deficiency of the cystine uptake limits the survival and growth of L1210 cells in vitro. The cystine uptake of one cloned cell line was characterized. The enhanced uptake of cystine in these cells was mainly mediated by a Na⁺-independent, saturable system and was potently inhibited by glutamate and some other anionic amino acids, but less by aspartate. Such activity of cystine uptake was not observed in the original L1210 cells. The results suggest that, upon adaptation in vitro, L1210 cells acquire a new cystine transport activity necessary for survival and growth in vitro.     

Effect of clofibrate on peroxisomal and mitochondrial beta-oxidation in chicken liver

The effect of a 0.25% clofibrate diet for 2 weeks on peroxisomal and mitochondrial beta-oxidation in chicken liver was studied. The activities of antimycin antimycin A-insensitive palmitoyl-CoA oxidation (peroxisomal beta-oxidation) and carnitine acetyltransferase increased about two-fold. The activities of palmitoyl-CoA-dependent O2 consumption (mitochondrial beta-oxidation) and carnitine palmitoyltransferase were also slightly activated by the administration of clofibrate, but not significant. Thus, clofibrate may be a typical drug which activates the peroxisomal beta-oxidation more than the mitochondrial one in various species. The effect of clofibrate on peroxisomal carnitine acetyltransferase was the same as that on the mitochondrial one in chicken liver. Serum lipids were not lowered, but hepatomegaly was observed in the present experiment with chicken.     

The unique action for bone metabolism of 1 alpha,25-(OH)2D3-26,23-lactone

[23 (S), 25 (R)]-1 alpha,25-Dihydroxyvitamin D3-26,23-lactone [( 23 (S),25 (R)]-1 alpha,25-(OH) 2D3-26,23-lactone) increased dose-dependently alkaline phosphatase activity in osteoblastic cells, clone MC3T3-E1, in medium containing 0.1% bovine serum albumin. The maximal stimulated enzyme activity per mg protein was 1.6-fold over that of control cultures at 250 pg/ml. The metabolite also increased collagen synthesis in a dose-related fashion. On the other hand, [23 (S),25 (R)]-1 alpha,25-(OH)2D3-26,23-lactone decreased slightly but significantly 45Ca mobilization, and blocked the resorptive action of 1 alpha,25-dihydroxyvitamin D3 but not that of parathyroid hormone, in mouse calvaria in organ culture. These results indicate that [23 (S),25 (R)]-1 alpha, 25-(OH)2D3-26,23-lactone stimulates the differentiation of osteoblasts and inhibits bone resorption in vitro.     

Isolation and characterization of the bacteriophage T4 tail-associated lysozyme.

Direct evidence has been obtained that the tail-associated lysozyme of bacteriophage T4 (tail-lysozyme) is gp5, which is a protein component of the hub of the baseplate. Tails were treated with 3 M guanidine hydrochloride containing 1% Triton X-100, and the tail-lysozyme was separated from other tail components by preparative isoelectric focusing electrophoresis as a peak with a pI of 8.4. The molecular weight as determined from sodium dodecyl sulfate electrophoresis was 42,000. The tail-lysozyme was unambiguously identified as gp5 when the position of the lysozyme was compared with that of gp5 of tube-baseplates from 5ts1/23amH11/eL1ainfected Escherichia coli cells by two-dimensional gel electrophoresis. The tail-lysozyme has N-acetylmuramidase activity and the same substrate specificity as gene e lysozyme; the optimum pH is around 5.8, about 1 pH unit lower than for the e lysozyme. We assume that the tail-lysozyme plays an essential role in locally digesting the peptidoglycan layer to let the tube penetrate into the periplasmic space. The tail-lysozyme is presumably also responsible for "lysis from without."     

Transient glucose intolerance during attacks of thyrotoxic periodic paralysis

In a 19-year-old Japanese male (case 1) with thyrotoxic periodic paralysis (TPP), an increase of plasma glucose concentration together with abnormally high levels of serum immunoreactive insulin (IRI) was observed preceding a spontaneous attack of paralysis. Therefore, the plasma glucose, glucagon, epinephrine, norepinephrine, serum IRI, growth hormone and cortisol levels, and the erythrocyte insulin receptors were measured in case 1 and a 40-year-old Japanese male (case 2) with TPP during attacks of paralysis induced by prolonged glucose loading. In case 1, the serum IRI concentration was elevated to the extraordinarily high level of 655.0 microU/ml at the beginning of paralysis, and at that time, the plasma glucose concentration was 147 mg/dl. However, when paralysis was not induced by a similar glucose loading during methimazole treatment, the serum IRI and plasma glucose levels at the corresponding time after glucose loading were 20.9 microU/ml and 87 mg/dl, respectively. Furthermore, the affinity of the erythrocyte insulin receptors was decreased during the attack. In case 2, plasma glucose and serum IRI concentrations were increased in accordance with the initiation of paralysis although the blood levels of hormones counteracting insulin were not significantly changed. These findings suggest that there is something interacting with the normal action of the insulin in the early phase of paralysis.