Isolation and characterization of the M.EaeI modification methylase.

The modification enzyme (M.EaeI) corresponding to the restriction endonuclease EaeI was partially purified from Enterobacter aerogenes PW201. The M.EaeI enzyme methylates the innermost cytosine residue in each strand of the family of related sequences that constitute the EaeI recognition site to give: 5'-Y-G-G-5mC-C-R-3' where 5mC is 5-methylcytosine. M.EaeI protects these sites against cleavage by HaeIII, and protects overlapping 5'-C-C-G-G-3' sites against cleavage by both HpaII and MspI.     

Deletions of the esterase D locus from a survey of 200 retinoblastoma patients

Summary Esterase D levels from 200 retinoblastoma patients have been measured in an attempt to identify individuals carrying deletions of chromosome region 13q14. In this series 75% had bilateral tumours and 23% were familial. Of nine patients identified as having low esterase D levels, five had not previously been diagnosed as deletion carriers. These observations demonstrate the benefit of screening retinoblastoma populations for esterase D deficiency.     

Mechanism of interferon action. Effect of double-stranded RNA and the 5'-O-monophosphate form of 2',5'-oligoadenylate on the inhibition of reovirus mRNA translation in vitro

The effect of reovirus double-stranded RNA (dsRNA) and 5'-O-monophosphate form of 2',5'-oligoadenylate (pA(2'p5'A)2) on the translation and degradation of reovirus messenger RNA and on protein phosphorylation was examined in extracts prepared from interferon-treated mouse L fibroblasts. The following results were obtained. 1) The enhanced degradation of reovirus [3H]mRNA observed in the presence of either dsRNA or the 5'-O-triphosphate form of 2',5'-oligoadenylate (pppA(2'p5'A)3) was completely blocked by pA(2'p5'A)2. 2) The dsRNA-dependent phosphorylation of protein P1 and the alpha subunit of eukaryotic initiation factor (eIF-2) depended in a similar manner upon the concentration of dsRNA and was optimal at low dsRNA concentrations (0.1 to 1 microgram/ml). However, high concentrations of dsRNA (greater than 100 micrograms/ml) drastically reduced the phosphorylation of both P1 and eIF-2 alpha. Neither P1 nor eIF-2 alpha phosphorylation was affected by either pA(2'p5'A)2 or pppA(2'p5'A)3. 3) The translation of reovirus mRNA in vitro was inhibited by the addition of either low concentrations of dsRNA or pppA(2'p5'A)3. Whereas pA(2'p5'A)2 completely reversed the pppA(2'p5'A)3-mediated inhibition of translation, the inhibition mediated by low concentrations of dsRNA was only partially reversed by pA(2'p5'A)2. Under conditions where the pppA-(2'p5'A)3mediated degradation of reovirus mRNA was blocked, the translation of reovirus mRNA was still inhibited by low but not by high concentrations of dsRNA in a manner that correlated with the activation of P1 and eIF-2 alpha phosphorylation. These results suggest that the pppA(2'p5'A)n-dependent ribonuclease is not required and that protein phosphorylation may indeed be sufficient for the dsRNA-dependent inhibition of reovirus mRNA translation in cell-free systems derived from interferon-treated mouse fibroblasts.     

Halalaimus algeriensis n. sp. (Nematoda) from the Sahara

During a trans-Saharan expedition in 1980 a number of samples were collected from stagnant and running fresh and slightly saline water bodies. One of them, collected from the Oued En-Namous in a small oasis yielded several interesting nematodes, among which was a newHalalaimus species described herein asHalalaimus algeriensis n. sp. It comes close toH. minusculus Tchesunov, 1978, but differs in tailshape, absence of males and habitat. It also resembles the marine speciesH. gracilis de Man, 1888, but differs in the relative length of the anterior setae, the absence of a lateral field and absence of males. The new species can be differentiated fromH. stammeri Schneider, 1940, the only fresh water species found hitherto, by its shorter body, more anterior and wider fovea, and the length and position of the anterior setae. The various juvenile stages can be separated on the basis of body length, foveal length and genital primordium.     

Characterization of the chicken erythrocyte anion exchange protein

The avian erythrocyte anion exchange protein (band 3), after labeling with [3H2]4,4'-diisothiocyanodihydrostilbene-2, 2'-disulfonic acid appears as a doublet of polypeptide chains with apparent Mr = 105,000 and 100,000 by sodium dodecyl sulfate gel electrophoresis. The structures of the two species are almost identical as determined by partial proteolysis. The copy number of band 3 molecules per chicken erythrocyte was determined to be 800,000 by quantitating the amount of [3H2]4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid covalently bound to the cell surface. A comparison of human and chicken band 3 has revealed differences in their structure. Chicken band 3 differs from the human polypeptide in isoelectric point and proteolytic patterns. Antisera raised against human and chicken band 3 do not cross-react, implying that the two sera do not recognize any common antigenic determinants. There is a 6.5-fold lower activity per cell in the rate of phosphate exchange in the chicken erythrocyte which can be entirely explained by the 1.5-fold decrease in copy number per cell and the increased size of the chicken erythrocyte. This would suggest that there is no difference in the enzyme turnover number between chicken and human band 3. A major functional difference resulting from the structural differences is the inability to bind glyceraldehyde-3-phosphate dehydrogenase, a function associated with the NH2 terminus of human band 3.     

The significance of reutilization of surfactant phosphatidylcholine

To assess the magnitude of reutilization of surfactant phosphatidylcholine, 68 3-day-old rabbits were injected intratracheally with a trace dose of [3H]choline-labeled surfactant mixed with [14C]palmitate-labeled synthetic dipalmitoylphosphatidylcholine. After timed kills we measured the total phosphatidylcholine associated counts/min in whole lung and alveolar wash and the specific activities of phosphatidylcholine in the alveolar wash, lamellar bodies, and microsomes isolated from the lung of each rabbit. Using a modification of the compartment analysis of Skinner et al. (Skinner, S. M., Clark, R. E., Baker, N., and Shipley, R. A. (1959) Am. J. Physiol. 196, 238-244), we found that surfactant phosphatidylcholine was reutilized with greater than 90% efficiency. The turnover time of the alveolar wash phosphatidylcholine was estimated to be 10.1 h and 9.3 h as measured by the 3H and 14C labels, respectively. From the ratios of alveolar wash-associated natural to synthetic phosphatidylcholine specific activities and from similar ratios obtained in 30 additional rabbits using [14C]choline-labeled natural surfactant and [3H]choline-labeled dipalmitoylphosphatidylcholine, we showed that phosphatidylcholine was reutilized intact rather than as component parts. Within 6 h of injection, the synthetic dipalmitoylphosphatidylcholine functioned metabolically as that administered in the form of natural surfactant.     

In Vivo Measurement of Indole-3-acetic Acid Decarboxylation in Aging Coleus Petiole Sections

The concentration of indoleacetic acid (IAA) in plant tissues is regulated, in part, by its rate of decarboxylation. However, the commonly used in vitro assays for IAA oxidase may not accurately reflect total in vivo decarboxylation rates. A method for measuring in vivo decarboxylation was utilized in which ¹⁴CO₂ is collected following uptake of [1-¹⁴C]IAA by excised tissue sections. After a 30-minute equilibration period, the evolution of ¹⁴CO₂ was found to follow an approximately linear course with respect to both time and tissue weight.

Decarboxylation rates were measured by this method in petiole sections of the Princeton clone of Coleus blumei Benth. Both the ¹⁴CO₂ evolved per milligram tissue and the percent of [1-¹⁴C]IAA uptake decarboxylated were highest in sections from the youngest petioles tested, and declined in the older tissue. Thin layer chromatography of acetonitrile extracts from the [1-¹⁴C]IAA-treated petioles showed a decreasing amount of free IAA and an increase at the retardation factor of indoleacetylaspartate in the older sections. The decreased decarboxylation rates in the older petioles may be attributable to a generally lower metabolic rate and increased protection of the IAA by conjugation.

     

Standardization of Radiorenography in Dehydrated and Rehydrated Primates Under Laboratory Conditions

Radiorenography with ^99mTo-labelled diethylenctriaminepcntacetic acid ([^99mTc]-DTPA) was performed on chacma baboons (Papio ursinus) and vervet monkeys (Cereopithecus pygerythus) to establish the effects of various states of hydration on the data obtained from the DTPA-renogram. The renogram parameters, which can be related to certain aspects of kidney function, varied significantly with the degree of hydration. It is therefore imperative for clinically directed animal research projects on the urinary system to standardise the experimental procedure for radiorenography. A dehydration of 6 h followed by an hour IU rehydration period using 200 ml of a 0.9% NaCI solution on baboons under thiopentone sodium anaesthetic, was found to be the most suitable procedure for radiorenographic investigations in this primate model.     

Synchronization of estrus in post-partum mares with progesterone and estradiol 17β

Thirty-six mares which foaled over a 10-day period were given 1 to 10 daily intramuscular injections of a combination of 150 mg. progesterone and 10 mg. estradiol 17β. The first injection was given within 18 hours after parturition. Because individual mares foaled on different dates during the 10 day period, commencement of treatment varied, but treatment for all mares ceased on the same day. Teasing and breeding began seven days after the final treatment. The mares were teased daily for 10 days and artifically inseminated every second day until ovulation occurred. The mean interval from the end of treatment to beginning of estrus was 9.4 days (range 7 to 14) and 33 of 26 mares (94.7%) ovulated 10 to 16 days after the final treatment. Both estrus and ovulation were effectively synchronized, resulting in a first estrus pregnancy rate of 80.6% (29 of 36).