The forever young gene encodes an oxidoreductase required for proper development of the Arabidopsis vegetative shoot apex

In plant development, leaf primordia are formed on the flanks of the shoot apical meristem in a highly predictable pattern. The cells that give rise to a primordium are sequestered from the apical meristem. Maintenance of the meristem requires that these cells be replaced by the addition of new cells. Despite the central role of these activities in development, the mechanism controlling and coordinating them is poorly understood. These processes have been characterized in the Arabidopsis mutant forever young (fey). The fey mutation results in a disruption of leaf positioning and meristem maintenance. The predicted FEY protein shares significant homology to a nodulin and limited homology to various reductases. It is proposed that FEY plays a role in communication in the shoot apex through the modification of a factor regulating meristem development.     

Analysis of the frequency of inheritance of transposed Ds elements in Arabidopsis after activation by a CaMV 35S promoter fusion to the Ac transposase gene

The Ac/Ds transposon system of maize shows low activity in Arabidopsis. However, fusion of the CaMV 35S promoter to the transposase gene (35S::TPase) increases the abundance of the single Ac mRNA encoded by Ac and increases the frequency of Ds excision. In the experiments reported here it is examined whether this high excision frequency is associated with efficient re-insertion of the transposon. This was measured by using a Ds that carried a hygromycin resistance gene (HPT) and was inserted within a streptomycin resistance gene (SPT). Excision of Ds therefore gives rise to streptomycin resistance, while hygromycin resistance is associated with the presence of a transposed Ds or with retention of the element at its original location. Self-fertilisation of most individuals heterozygous for Ds and 35S::TPase produced many streptomycin-resistant (strep^r) progeny, but in many of these families a small proportion of strep^r seedlings were also resistant to hygromycin (hyg^r). Nevertheless, 70% of families tested did give rise to at least one strep^r, hyg^r seedling, and over 90% of these individuals carried a transposed Ds. In contrast, the Ac promoter fusion to the transposase gene (Ac::TPase) produced fewer strep^rhyg^r progeny, and only 53% of these carried a transposed Ds. However, a higher proportion of the strep^r seedlings were also hyg^r than after activation by 35S::TPase. We also examined the genotype of strep^r, hyg^r seedlings and demonstrated that after activation by 35S::TPase many of these were homozygous for the transposed Ds, while this did not occur after activation by Ac::TPase. From these and other data we conclude that excisions driven by 35S::TPase usually occur prior to floral development, and that although a low proportion of strep^r progeny plants inherit a transposed Ds, those that do can be efficiently selected with an antibiotic resistance gene contained within the element. Our data have important implications for transposon tagging strategies in transgenic plants and these are discussed.     

Chromosome 14 and late-onset familial Alzheimer disease (FAD)

Familial Alzheimer disease (FAD) is genetically heterogeneous. Two loci responsible for early-onset FAD have been identified: the amyloid precursor protein gene on chromosome 21 and the as-yet-unidentified locus on chromosome 14. The genetics of late-onset FAD is unresolved. Maximum-likelihood, affected-pedigree-member (APM), and sib-pair analyses were used, in 49 families with a mean age at onset ≥60 years, to determine whether the chromosome 14 locus is responsible for late-onset FAD. The markers used were D14S53, D14S43, and D14S52. The LOD score method was used to test for linkage of late-onset FAD to the chromosome 14 markers, under three different models: age-dependent penetrance, an affected-only analysis, and age-dependent penetrance with allowance for possible age-dependent sporadic cases. No evidence for linkage was obtained under any of these conditions for the late-onset kindreds, and strong evidence against linkage (LOD score ≤ –2.0) to this region was obtained. Heterogeneity tests of the LOD score results for the combined group of families (early onset, Volga Germans, and late onset) favored the hypothesis of linkage to chromosome 14 with genetic heterogeneity. The positive results are primarily from early-onset families. APM analysis gave significant evidence for linkage of D14S43 and D14S52 to FAD in early-onset kindreds (P < .02). No evidence for linkage was found for the entire late-onset family group. Significant evidence for linkage to D14S52, however, was found for a subgroup of families of intermediate age at onset (mean age at onset ≥60 years and <70 years). These results indicate that the chromosome 14 locus is not responsible for Alzheimer disease in most late-onset FAD kindreds but could play a role in a subset of these kindreds.     

Framework for validation and implementation of in vitro toxicity tests

Summary The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technologic developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial, and regulatory communities, is recommended. Test validation acceptance is contingent on broad buy-in by disparate groups in the scientific community—academics, industry, and government. This is best achieved by early and frequent communication among parties and agreement on common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction, and refinement alternatives in toxicity testing.     

Limits to Catalysis by Ribonuclease A

Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of the P-O(5') bond in RNA. Although this enzyme has been the object of much landmark work in bioorganic chemistry, the nature of its rate-limiting transition state and its catalytic rate enhancement had been unknown. Here, the value of k(cat)/K(m) for the cleavage of UpA by wild-type RNase A was found to be inversely related to the concentration of added glycerol. In contrast, the values of k(cat)/K(m) for the cleavage of UpA by a sluggish mutant of RNase A and the cleavage of the poor substrate UpOC(6)H(4)-p-NO(2) by wild-type RNase A were found to be independent of glycerol concentration. Yet, UpA cleavage by the wild-type and mutant enzymes was found to have the same dependence on sucrose concentration, indicating that catalysis of UpA cleavage by RNase A is limited by desolvation. The rate of UpA cleavage by RNase A is maximal at pH 6.0, where k(cat) = 1.4 × 10(3) s(-1) and k(cat)/K(m) = 2.3 × 10(6) M(-1)s(-1) at 25°C. At pH 6.0 and 25°C, the uncatalyzed rate of [5,6-(3)H]Up[3,5,8-(3)H]A cleavage was found to be k(uncat) = 5 × 10(-9) s(-1) (t(1/2) = 4 years). Thus, RNase A enhances the rate of UpA cleavage by 3 × 10(11)-fold by binding to the transition state for P-O(5') bond cleavage with a dissociation constant of <2 × 10(-15) M.     

Role of the conserved C-repeat region of the M protein of Streptococcus pyogenes

The surface-located M protein functions to protect Streptococcus pyogenes (the group A streptococcus) from phagocytosis by polymorphonuclear leukocytes. It has been suggested that this protection results from the ability of M protein to bind factor H, a serum protein that can inhibit the activation of complement. Among different serological variants of M protein, the C-repeat domain is highly conserved and is exposed on the bacterial surface. This domain has been implicated in binding to complement factor H and in M-protein-mediated adherence of streptococci to human keratinocytes in the cutaneous epithelium. In this study, we constructed an S. pyogenes mutant strain which expresses an M6 protein from which the entire C-repeat domain was deleted. As predicted, this mutant did not adhere well to human keratinocytes and was unable to bind to factor H. Unexpectedly, the mutant was able to survive and multiply in human blood. Therefore, while the binding of factor H and the facilitation of adherence to keratinocytes appear to involve recognition of the C-repeat domain, a region of the M-protein molecule distinct from the C-repeat domain confers upon S. pyogenes its ability to resist phagocytosis.     

Inhibition of polar calcium movement and gravitropism in roots treated with auxin-transport inhibitors

Primary roots of maize (Zea mays L.) and pea (Pisum sativum L.) exhibit strong positive gravitropism. In both species, gravistimulation induces polar movement of calcium across the root tip from the upper side to the lower side. Roots of onion (Allium cepa L.) are not responsive to gravity and gravistimulation induces little or no polar movement of calcium across the root tip. Treatment of maize or pea roots with inhibitors of auxin transport (morphactin, naphthylphthalamic acid, 2,3,5-triiodobenzoic acid) prevents both gravitropism and gravity-induced polar movement of calcium across the root tip. The results indicate that calcium movement and auxin movement are closely linked in roots and that gravity-induced redistribution of calcium across the root cap may play an important role in the development of gravitropic curvature.Abbreviations 9-HFCA 9-hydroxyfluorenecarboxylic acid - NPA naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid - IAA indole-3-acetic acid     

Adenylate cyclase and cyclic nucleotide phosphodiesterases in the developing rat liver

A potential regulatory role for the cyclic nucleotides during liver morphogenesis will be better understood as the development of various components of the cyclic nucleotide system are characterized. Accordingly, adenylate cyclase response to glucagon and 5′-guanylimidodiphosphate (Gpp(NH)p) and the specific activities, cellular distributions, and kinetic constants (V and K_m) of the cyclic AMP and cyclic GMP phosphodiesterases were determined at variuos stages of rat liver development. These results show (1) a period of increasing sensitivity of rat liver adenylate cyclase to glucagon at a time when sensitivity to NaF and Gpp(NH)p remains unchanged, and (2) increased responsiveness to glucagon plus Gpp(NH)p which is dependent upon the degree of glucagon sensitivity. It is concluded that the guanul nucleotide regulatory site is a functional part of adenylate cyclase very early in liver development and that the development of glucagon sensitivity is more probably limited by the developmet of glucagon receptors. Two forms of each phosphodiesterase (high and low K_m) were found throughout, except that low K_m cyclic GMP phosphodiesterase could not be demonstrated in the embryo. No significant change with age was found for the K_m or V of any of the enzyme forms. The ratio of soluble: particulate cyclic AMP phosphodiesterase decreased with age, whereas no change in the ration for cyclic GMP phosphodiesterase was observed. Specific activities of each enzyme from were highest in the perinatal period and decreased with age. The changes in phosphodiesterase specific activities paralled changes in guanylate and adenylate cyclase activities, which argues against a selective regulatory role for phosphodiesterase in modulating cyclic nucleotide influences during liver morphogenesis.     

Reproductive patterns in seventeen species of warmwater fishes in a Missouri River reservoir

Synopsis The timing of ovarian maturation and spawning of 17 warmwater fish species in Lake Oahe (South and North Dakota) was estimated from changes in the mean ovary indices (ratios of ovary weight to fish length). The onset of vitellogenesis varied within species (up to 2 months). Maturation of the ova took from 7.5 to 10 months, depending on species. Annual variations in the mean date of peak spawning of individual species during 1964–1971 were usually less than a week. There was little overlap of the annual mean peak spawning dates of the 17 species, and an established sequence of spawning among species was shown. A relatively high incidence of atresia in the shovelnose sturgeon, northern pike, and carp indicated that these species had apparently not yet adapted to the altered and variable spawning conditions in this reservoir. Regularity of spawning would seem to provide the best chance for spawning success in variable environments such as Lake Oahe.