Forskolin and 3-Isobutyl-l-Methylxanthine Increase Basal and Sodium Nitroprusside-Elevated Cyclic GMP Levels in Adult Guinea-Pig Cerebellar Slices

Abstract: In this study, the interaction between 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) in [³H]adenine-or [³H]-guanine-prelabelled adult guinea-pig cerebellar slices was investigated. Basal levels of [³H]cGMP were enhanced by forskolin, although no plateau was reached over the concentration range tested (0.1-100 μM). However, forskolin elicited a concentration-dependent, saturable potentiation of sodium nitroprusside (SNP)-stimulated [³H]cGMP accumulation (forskolin EC₅₀ value of 0.98 β 0.23 μM; 10 μM forskolin produced a 1.8 β 0.3-fold potentiation of the SNP response at 2.5 min). The forskolin potentiation was observed at all concentrations of SNP tested (0.001-10 mM). forskolin also elicited a large stimulation of [³H]-cAMP in [³H]adenine-prelabelled guinea-pig cerebellar slices; however, 1,9-dideoxyforskolin failed to elicit either a [³H]cAMP response or a potentiation of the SNP-induced [³H]cGMP response at concentrations up to 100 μM. Pretreatment with oxyhaemoglobin (50 μM) inhibited the response to SNP (1 mM) and forskolin (10 μM), as well as the response evoked by the combination of SNP and forskolih. A^G-Nitro-l -arginine (100 μM) inhibited the response to forskolin alone, but did not change the response to SNP or the potentiation induced by forskolin on SNP-induced [³H]cGMP levels. The protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7; 100 μM), staurosporine (10 μM), polymyxin B (100 μM), and Ro 31-8220 (10 μM) had no effect on the [³H]cGMP response to either SNP or the combination of SNP plus forskolin. N⁶,2′-Dibutyryl cAMP, at concentrations up to 10 mM, was also without effect on [³H]cGMP levels induced by SNP. 3-lso-butyl-1-methylxanthine reproduced the effect of forskolin on SNP-induced [³H]cGMP levels, but a less-than-additive effect was observed when the response to SNP was studied in the presence of forskolin and 3-isobutyl-1-methylxanthine. Taken together, these results infer that crosstalk between cyclic nucleotides takes place in guinea-pig cerebellar slices, and that cAMP may regulate cGMP-mediated responses in this tissue.     

D-Amino acids in chronic renal failure and the effects of dialysis and urinary losses

Summary Total D-amino acids were measured in plasma for 20 non-dialysed patients (creatinine clearance < 12 ml/minute), 20 on CAPD, 20 on haemodialysis and 20 normals. Plasma D-tyrosine and D-phenylalanine were measured in 8 of each group by HPLC. Total D-amino acids, D-tyrosine and D-phenylalanine were significantly greater for patients than normals. D-amino acids and D-tyrosine correlated with creatinine and were decreased during HD. During dialysis, the mean losses for D-tyrosine and D-phenylalanine were similar, about 0.2 mg/CAPD exchange and 3 mg/4 hour haemodialysis (i.e. 2% of the total amino acid, as in plasma). Clearance was unaffected by stereochemical configuration. Urinary losses/24 hour in the non-dialysed patients were 0.35 mg D-tyrosine and 0.25 mg D-phenylalanine. Clearance for D-phenylalanine was greater than for the L-enantiomer. Increases in D-amino acids in renal failure are probably due to depletion of D-amino acid oxidase, but may be enhanced by a D-amino acid rich diet, peptide antibiotics and D-amino acid oxidase inhibiting drugs and metabolites. Possible toxic effects need further investigation.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Cryopreservation of immature spring wheat zygotic embryos using an abscisic acid pretreatment

Spring wheat (Triticum aestivum L.) zygotic embryos were successfully cryopreserved, without the addition of exogenous cryoprotectants, using only an abscisic acid (ABA) pretreatment. Optimum survival was obtained when embryos were cultured in vitro for 10 days on semisolid Murashige and Skoog (MS) nutrient medium supplemented with 0.5 mg/L (±) ABA prior to cryopreservation. The embryos resumed growth within three days when returned to MS medium devoid of ABA but containing 2mg/L 2,4-dichlorophenoxyacetic acid. The embryogenic calli produced from these embryos exhibited normal plant regeneration on auxin-free media. Changes in dw/fw ratio, as well as the esterified fatty acid and sucrose concentrations correlated positively with the development of tolerance to cryopreservation.NRCC Publication No. 33519     

An alternate model of thyroid cytology: practical not perfect

A sample of 384 thyroid cytology specimens prepared by cytospin over a 2.5-year period was classified by original report into inadequate, non-neoplastic and suspicious of neoplasia or worse. This was then compared with subsequent histology. The resulting data showed an inadequacy rate of 33%, a sensitivity of 55%, a specificity of 59%, a positive predictive value of 64% and a negative predictive value of 93%. On review of the cytology, in knowledge of the subsequent histology, the maximum achievable results were determined to have a positive predictive value of 79% and a negative predictive value of 97%. No clinically significant adverse event was detected.     

Mutational analysis of the RecA protein L1 region identifies this area as a probable part of the co-protease substrate binding site

Previous mutational analysis of the L1 region of the RecA protein suggested that Gly-157 and Glu-158 are 'hot-spots' for the occurrence of constitutive LexA co-protease mutants (coprt^c). In the present study, we clearly establish that position 157 is a hot-spot for the occurrence of such mutants, as 12 of 14 and 10 of 14 substitutions result in this phenotype for UmuD and LexA cleavage respectively. The frequency of such mutations at position 158 is somewhat lower, 8 of 13 and 5 of 13 for UmuD and LexA respectively. Comparison of the UmuD vs. LexA co-protease activity for all single mutants with substitutions at positions 154, 155, 156, 157 and 158 (47 in total) reveals that, although there is good agreement among most mutants regarding their ability to cleave both LexA and UmuD, there are two in particular (Glu-154→Asp and Glu-154→Gln) that show a clear preference for cleavage of UmuD. We also show that three second-site mutations that completely suppress coprt^c activity toward LexA have little or no effect on the coprt^c activity of the primary mutant toward UmuD. In addition, we observe a high frequency of second-site suppressor mutations, suggesting a functional interaction among side-chains in this region. Together, these results support the idea that the L1 region of RecA makes up part of the co-protease substrate-binding site.     

Endotoxin shock: prevented by naloxone in intact but not hypophysectomized rats

Previous studies established that naloxone reverses hypotension in endotoxin, hemorrhagic, and spinal shock. We studied endotoxin shock in hypophysectomized (Hx) rats, which have little circulating beta-endorphin. Hx or intact rats received surgically implanted jugular catheters for drug injection and aortic catheters for arterial blood pressure (MAP) recording. On the second day after implantation, rats were pretreated with either naloxone or saline. Two minutes later each rat received endotoxin. Following endotoxin, all rats showed a brief biphasic hypertensive-hypotensive response followed by stabilization of MAP near baseline. Within 20 min, all Hx rats, regardless of pretreatment, and the saline-treated intact rats, showed progressive hypotension (P less than 0.005). Only the naloxone-pretreated intact rats maintained a stable MAP. Plasma endorphin measured at 20 min was undetectable in Hx rats in contrast to intact rats (P less than 0.001); plasma corticosterone levels were likewise suppressed in the Hx rats (P less than 0.01). Thus (1) naloxone protected only the rats with an intact pituitary-adrenal-sympathetic system, and (2) pituitary endorphin is not required to generate endotoxin shock in hypophysectomized rats.     

Heterogeneity of epithelial cells in the human thymus

Summary To evaluate interrelationships among epithelial cells, and between morphology and function in the microenvironment, we studied the ultrastructural morphology of epithelial cells in sections of human thymus from donors aged 2 months to 31 years. Six types of epithelial cells were observed: subcapsular-perivascular (type 1); pale (type 2); intermediate (type 3); dark (type 4); undifferentiated (type 5); and large-medullary (type 6). Cells of types 2, 3 and 4 were found throughout the organ. The type-2 to -4 epithelial cells may represent various stages in a differentiation process. In this, type-2 cells are very active and type-4 cells are possibly degenerating elements. Type-4 cells can also contribute to Hassall's corpuscles. Type-5 cells were located mainly in the cortico-medullary region and showed the morphological characteristics of undifferentiated elements. Type-6 cells were located exclusively in the medulla and displayed characteristics of cellular activity. Small Hassall's corpuscles consisted of type-6 epithelial cells; in larger corpuscles many nuclei of type-6 cells were found. Cells of types 2 and 6 contained tubular structures (diameter approximately 20 nm).Concerning the function of thymus epithelial cells, the features associated with protein synthesis observed in cellular types 2 and 6 make them likely candidates for humoral factor-producing and/or secreting elements. In addition, type-2 and -3 cells in the cortex appear to contribute to a special pattern of epithelium-lymphocyte interaction (thymic nurse cells), as demonstrated by the intracytoplasmic location of lymphocytes in the epithelial cells. The various steps in intrathymic T-cell maturation occur at locations in a microenvironment composed of morphologically distinct epithelial cells.     

Mapping of nucleoside phosphorylase (Np-1) and esterase 10 (Es-10) on mouse chromosome 14

A method for detecting two alleles at Np-1 (nucleoside phosphorylase) and three alleles at Es-10 (esterase 10) from mouse blood by cellulose acetate electrophoresis is described. The allelic constitution at these loci for 44 inbred strains and stocks was determined. The location of Np-1 on chromosome 14 was established by backcross experiments in which alleles at Np-1 and Robertsonian translocations were segregating. Es-10 was shown to be linked to Np-1, and the following genetic map of Chr 14 was constructed: centromere-(8.9±4.0 cM)-[Np-1, Wc]-(10.2±1.9 cM)-Es-10-(15.5±3.7 cM)-s. The homologous human loci, NP and ES-D, are not linked.This work was supported by Contract E(11-1)-3267 with the Energy Research and Development Administration, by Contracts NO1-ES4-2156 and NO1-ES4-2159 with the National Institute of Environmental Health Sciences, and by Grants GM 19656 and GM 20919 from the National Institute of General Medical Sciences. D. A. K. was a participant in the 1975 Summer Program for College, Graduate, and Medical Students, which was supported, in part, by the Clark Foundation. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Reversible (G0) and nonreadily reversible (Q) noncycling cells in human peripheral blood. Immunological, structural, and biological characterization

PHA-stimulated human lymphocytes (normal-resting-proliferating) at 0, 24, 48, 72, and 144 h were studied with Acridine Orange (AO) staining. By viable cell sorting, by subsequent subculturing, and by the use of biochemical, biophysical, and immunological assays, not only have the G0 resting and G1 (cycling) cell cycle phases been objectively characterized, but a separate subpopulation of quiescent cells that are functionally viable and deeply committed to nonproliferation, the Q cells, has been identified. Multiparameter cytofluorimetric analysis, methyl14C-thymidine incorporation, automated image analysis, and mitogen stimulation studies have shown that the "Q" cell, compared to the "G0" resting but easily recruitable cell, exhibits quite lower red and green AO emission, possesses 2c to 4c DNA content (rather than only 2c), has a higher average optical density, and is either nonrecruitable or recruitable-with-difficulty in PHA-stimulated lymphocyte cultures.