Families grieving together: Integrating the loss of a child through ongoing relational connections.

This study explored the relational dimensions of grieving within the family unit. Three families bereaved of a child, participated. Using the Qualitative Action-Project Method, individual and joint interviews were conducted with family members. Data analysis illuminated family grieving processes and demonstrated that grieving was an interactive process with individual, dyadic, multi-adic, and community levels of processing. The family grieving process included intentionality in grieving together and separately, recognition of differing grieving styles, and meaning came through the incorporation of ongoing rituals and remembrances. A finding emerged of family connection facilitated through an ongoing, shared, continuing bond with the deceased child.

Auxin signaling: derepression through regulated proteolysis.

Auxins are a class of phytohormones implicated in virtually every aspect of plant growth and development. Many early plant responses to auxin are apparently mediated by members of a family of Aux/IAA proteins that dimerize with and inhibit members of the auxin response factor (ARF) family of transcription factors. Aux/IAA proteins are unstable, and their degradation is triggered by a ubiquitin-protein ligase that is regulated by modification with a ubiquitin-related protein. Recent genetic and biochemical evidence indicates that auxin accelerates the degradation of the already short-lived Aux/IAA proteins to derepress transcription by ARF proteins. Several pieces of the auxin-signaling puzzle remain to be assembled, including the proteins that initially bind auxin, the proteins that convey this signal to the protein degradation machinery, and the targets of the transcriptional derepression.

ILR1, an amidohydrolase that releases active indole-3-acetic acid from conjugates.

In plants, the growth regulator indole-3-acetic acid (IAA) is found both free and conjugated to a variety of amino acids, peptides, and carbohydrates. IAA conjugated to leucine has effects in Arabidopsis thaliana similar to those of free IAA. The ilr1 mutant is insensitive to exogenous IAA-Leu and was used to positionally clone the Arabidopsis ILR1 gene. ILR1 encodes a 48-kilodalton protein that cleaves IAA-amino acid conjugates in vitro and is homologous to bacterial amidohydrolase enzymes. DNA sequences similar to that of ILR1 are found in other plant species.

Redundancy as a way of life - IAA metabolism.

Plants have evolved elaborate systems for regulating cellular levels of indole-3-acetic acid (IAA). The redundancy of this network has complicated the elucidation of IAA metabolism, but molecular genetic studies and precise analytical methods have begun to expose the circuitry. It is now clear that plants synthesize, inactivate and catabolize IAA by multiple pathways, and multiple genes can encode a particular enzyme within a pathway. A number of these genes are now cloned, which greatly facilitates the future dissection of IAA metabolism.

Genetic analysis of indole-3-butyric acid responses in Arabidopsis thaliana reveals four mutant classes.

Indole-3-butyric acid (IBA) is widely used in agriculture because it induces rooting. To better understand the in vivo role of this endogenous auxin, we have identified 14 Arabidopsis mutants that are resistant to the inhibitory effects of IBA on root elongation, but that remain sensitive to the more abundant auxin indole-3-acetic acid (IAA). These mutants have defects in various IBA-mediated responses, which allowed us to group them into four phenotypic classes. Developmental defects in the absence of exogenous sucrose suggest that some of these mutants are impaired in peroxisomal fatty acid chain shortening, implying that the conversion of IBA to IAA is also disrupted. Other mutants appear to have normal peroxisomal function; some of these may be defective in IBA transport, signaling, or response. Recombination mapping indicates that these mutants represent at least nine novel loci in Arabidopsis. The gene defective in one of the mutants was identified using a positional approach and encodes PEX5, which acts in the import of most peroxisomal matrix proteins. These results indicate that in Arabidopsis thaliana, IBA acts, at least in part, via its conversion to IAA.

Cloning and characterization of the Arabidopsis thaliana lupeol synthase gene.

A 2274 bp Arabidopsis thaliana cDNA was isolated that encodes a protein 57% identical to cycloartenol synthase from the same organism. The expressed recombinant protein encodes lupeol synthase, which converts oxidosqualene to the triterpene lupeol as the major product. Lupeol synthase is a multifunctional enzyme that forms other triterpene alcohols, including beta-amyrin, as minor products. Sequence analysis suggests that lupeol synthase diverged from cycloartenol synthase after plants diverged from fungi and animals. This evolutionary order is the reason that fungi and animals do not make lupeol.

Mechanical evaluation of cross-link designs in rigid pedicle screw systems.

STUDY DESIGN: This study was designed to evaluate the biomechanical performance of 5 different cross-link brands to determine which design characteristics are biomechanically desirable. METHODS: The Cotrel-Dubousset, Isola, Puno Winter Byrd, Rogozinski, and Texas Scottish Rite Hospital systems were assembled to vertebral models according to the manufacturer's specifications. Three constructs were tested for each brand of instrumentation: without cross-links, with one cross-link, and with two cross-links. Four modes of loading: axial, torsional, flexion-extension, and lateral-flexion were used. Load-displacement curves were plotted. The stiffness was calculated from the slope of these curves. OBJECTIVES: Five different rigid pedicle screw systems were tested to determine: 1) what are the characteristics of cross-link design that are most effective in limiting torsional motion; 2) whether two cross-links are more effective than one; and 3) whether cross-linkage increases the construct stiffness in lateral bending. SUMMARY OF BACKGROUND DATA: Cross-linkage has been shown to increase the torsional stiffness of rod and screw constructs. Increased construct stiffness has been correlated with higher fusion rates. RESULTS: Increases in axial, flexion-extension, or lateral-flexion stiffness, with the addition of one or two cross-links, were not statistically significant. In torsional loading, increases in stiffness within brands were statistically significant in every case. The average increase was 44% with one added cross-link and 26% with two. The magnitude of the increase in torsional stiffness was compared with the cross-sectional area of the respective cross-link. Greater stiffness correlated with larger cross-sectional area (r = 0.81 for one cross-link, and r = 0.60 for two). CONCLUSION: The use of cross-linkage in spinal fusion increases torsional stiffness in pedicle screw and hook constructs. This study 1) confirmed the effectiveness of cross-linkage in limiting torsional motion and showed the superiority of two cross-links to one cross-link in limiting torsional motion, 2) showed that increase of torsional stiffness of a cross-linked construct is proportional to the cross-sectional area of the cross-link, and 3) demonstrated that cross-links do not increases stiffness in the lateral flexion mode.

Anterior instrumentation of the thoracolumbar spine. A biomechanical comparison.

STUDY DESIGN: To evaluate the fatigue strength and stiffness of four anterior thoracolumbar fixation devices using a corpectomy model without load-sharing bone graft to test the devices under the worst case scenario of instability. OBJECTIVES: To gain a more thorough understanding of the biomechanical qualities of anterior fixation devices to improve clinical application and design. SUMMARY OF BACKGROUND DATA: For many surgeons, the anterior approach has become the treatment of choice for patients with compression of the spinal cord, whether it is caused by trauma, tumor, or infection. When stabilization is needed, anterior fixation devices have been advocated for many years to avoid the additional approach required for posterior fixation. Many of these devices, however, have an unacceptably high rate of hardware failure. Recently, several new devices for anterior fixation have been marketed with purported advantages in fatigue life and ease of use. METHODS: Four implants, the Synthes Anterior Thoracolumbar Locking Plate, the Kaneda device, a Texas Scottish Rite Hospital anterior construct, and the Z-Plate were attached to vertebral models and tested for stiffness in multiple planes on a modified Materials Testing System machine. They then were fatigued to failure on an Instron testing machine. RESULTS: The Anterior Thoracolumbar Locking Plate was the stiffest in axial compression, lateral flexion, and torsion. The Texas Scottish Rite Hospital anterior construct was the least stiff in flexion-extension, with no significant differences in the stiffness of the anterior thoracolumbar locking plate, that of the Kaneda device, and that of the Z-Plate. Fatigue life exceeded 80,000 cycles for the anterior thoracolumbar locking plate and averaged 26,472 cycles for the Z-Plate, 6915 cycles for the Teas Scottish Rite Hospital construct, and 4419 cycles for the Kaneda device. CONCLUSIONS: The significantly greater fatigue life of the Anterior Thoracolumbar Locking Plate and the Z-Plate may predict a lower incidence of hardware failure than with previous anterior devices. This has been confirmed in preliminary clinical studies with the Z-Plate. Further clinical studies are needed to show if these lower failure rates will continue over a long-term period.

Hypersensitivity to heavy water: a new conditional phenotype.

Wild-type strains of the yeast S. cerevisiae can grow on media containing 90% D2O. Using chemical mutagenesis we obtained a number of strains that grow on H2O-containing media but not on otherwise identical media containing 90% D2O. The frequency of these D2O-sensitive (d) mutants is comparable to the frequency of conventional temperature-sensitive (ts) mutants in the same mutagenized sample, and the ds mutations are distributed over a large number of complementation groups. Furthermore, most ds mutants do not display other conditional phenotypes, such as heat, cold, or osmotic sensitivity. Conversely, of 17 cell division cycle ts mutants tested, only 2 are also ds. Thus, the ds technique should be useful for producing conditional mutations in genes that are not amenable to the ts and cs approaches, and also for generating alternative conditional (ds) alleles in many other genes. In addition, the ds technique should make it possible to generate conditional (ds) mutants in homeothermic animals, thereby extending the advantages of conditional phenotypes to mammalian and avian genetics.

A library of Arabidopsis 35S-cDNA lines for identifying novel mutants.

We have developed a system to over-express or co-suppress random cDNAs in Arabidopsis thaliana upon Agrobacterium tumefaciens-mediated transformation. We constructed a binary vector containing a novel Arabidopsis cDNA library driven by the cauliflower mosaic virus (CaMV) 35S promoter. The vector, 35SpBARN, offers in terra selection with glufosinate ammonium (BASTA) and the ability to identify the cDNA insert using PCR with flanking primers. We introduced this overexpression library into Arabidopsis and selected over 30,000 transformants. A random sample of 50 T1 plants was analyzed to determine the quality of the cDNA library in planta. About 90% of T1 plants in the collection have inserts, the average insert size is ca. 1.1 kb, and ca. 43% of these inserts appear to encode full-length proteins. T1 plants were screened for visible abnormalities, and one mutant, V5, was chosen for further study. This mutant displays a pale green phenotype, and its transgene contains a partial petH cDNA encoding chloroplast ferredoxin-NADP+ reductase (EC 1.18.1.2). This construct co-suppresses the endogenous petH transcript. We recapitulated the mutant phenotype by expressing either the full-length or truncated petH cDNA from the CaMV 35S promoter in wild-type Arabidopsis. Our results indicate that co-suppressing endogenous genes can cause dominant phenotypes as expected. As we have also used the 35SpBARN vector to successfully over-express other transcripts in planta, we predict that this system will be generally useful for identifying genes that yield phenotypes upon over-expression as well.

Differential regulation of an auxin-producing nitrilase gene family in Arabidopsis thaliana.

Nitrilases (nitrile aminohydrolase, EC 3.5.5.1) convert nitriles to carboxylic acids. We report the cloning, characterization, and expression patterns of four Arabidopsis thaliana nitrilase genes (NIT1-4), one of which was previously described [Bartling, D., Seedorf, M., Mithöfer, A. & Weiler, E. W. (1992) Eur. J. Biochem. 205, 417-424]. The nitrilase genes encode very similar proteins that hydrolyze indole-3-acetonitrile to the phytohormone indole-3-acetic acid in vitro, and three of the four genes are tandemly arranged on chromosome III. Northern analysis using gene-specific probes and analysis of transgenic plants containing promoter-reporter gene fusions indicate that the four genes are differentially regulated. NIT2 expression is specifically induced around lesions caused by bacterial pathogen infiltration. The sites of nitrilase expression may represent sites of auxin biosynthesis in A. thaliana.

Discharge patterns of motility-regulating neurons projecting in the lumbar splanchnic nerves to visceral stimuli in spinal cats.

Reflexes in visceral preganglionic motility-regulating (MR) neurons which project in the lumbar splanchnic nerves were investigated in acutely spinalized cats. Some neurons were analyzed before and after spinalization. The stimuli used were mechanical stimulation of mucosal skin of the anus and of perianal (perigenital) hairy skin, and distension and contraction of urinary bladder and colon. Most MR neurons exhibited a reflex pattern which consists of the following components: excitation upon bladder distension, inhibition or no effect upon colon distension and excitation (or, rarely, no effect) upon anal stimulation. This is the reflex pattern of MR1 neurons. Some neurons were excited by anal stimulation but not affected from the colon and urinary bladder. Some were inhibited by anal and perianal stimulation but otherwise exhibited the reflex patterns of the MR1 neurons. Analysis of the reflexes before and after spinalization showed that, in particular, inhibition elicited by anal, perianal and bladder stimulation was abolished; inhibition elicited from the colon was enhanced after spinalization. It is concluded that the reflexes elicited in preganglionic lumbar visceral neurons by the natural stimuli probably use spinal pathways, with the afferent input occurring at the sacral spinal cord. These spinal reflex pathways are probably controlled by descending inhibitory and excitatory spinal systems from the supraspinal neuraxis.

The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis.

Three of the four yeast ubiquitin genes encode hybrid proteins which are cleaved to yield ubiquitin and previously unidentified ribosomal proteins. The transient association between ubiquitin and these proteins promotes their incorporation into nascent ribosomes and is required for efficient ribosome biogenesis. These results suggest a novel 'chaperone' function for ubiquitin, in which its covalent association with other proteins promotes the formation of specific cellular structures.

The recognition component of the N-end rule pathway.

The N-end rule-based degradation signal, which targets a protein for ubiquitin-dependent proteolysis, comprises a destabilizing amino-terminal residue and a specific internal lysine residue. We report the isolation and functional analysis of a gene (UBR1) for the N-end recognizing protein of the yeast Saccharomyces cerevisiae. UBR1 encodes a approximately 225 kd protein with no significant sequence similarities to other known proteins. Null ubr1 mutants are viable but are unable to degrade the substrates of the N-end rule pathway. These mutants are partially defective in sporulation and grow slightly more slowly than their wild-type counterparts. The UBR1 protein specifically binds in vitro to proteins bearing amino-terminal residues that are destabilizing according to the N-end rule, but does not bind to otherwise identical proteins bearing stabilizing amino-terminal residues.

A gain-of-function mutation in IAA28 suppresses lateral root development.

The phytohormone auxin is important in many aspects of plant development. We have isolated an auxin-resistant Arabidopsis mutant, iaa28-1, that is severely defective in lateral root formation and that has diminished adult size and decreased apical dominance. The iaa28-1 mutant is resistant to inhibition of root elongation by auxin, cytokinin, and ethylene, but it responds normally to other phytohormones. We identified the gene defective in the iaa28-1 mutant by using a map-based positional approach and found it to encode a previously uncharacterized member of the Aux/IAA gene family. IAA28 is preferentially expressed in roots and inflorescence stems, and in contrast to other Aux/IAA genes, IAA28 transcription is not induced by exogenous auxin. Studies of the gain-of-function iaa28-1 mutant suggest that IAA28 normally represses transcription, perhaps of genes that promote lateral root initiation in response to auxin signals.

Behavior and brain neurotransmitters: correlations in different strains of mice.

Correlations of behavioral patterns in a social setting with catecholamines, serotonin, and several metabolites and precursors in three brain regions were examined in the DeFries H2, C1, and L1 strains of mice. In Experiment I, behavioral observations were recorded for two 15-min sessions in same-sex, same-strain pairs at about 65 days of age. In Experiment II, sex and strain groups were subdivided into 4% and 24% protein diet groups about 1 week before a second set of behavioral observations at about 120 days of age. Brain tissue content of neurotransmitters, precursors, and metabolites was determined by high-performance liquid chromatography after the second set of observations. Significant multivariate strain differences were shown for behavioral variables (both experiments) as well as concentrations of various neurochemicals. Strain H2 showed relatively high levels of locomotion, while rearing and social investigation were high in strain C1 and self-grooming in strain L1. Significant neurochemical differences were found in the following sets of variables: dopamine variables in the cortex, norepinephrine variables and serotonin variables in the combined diencephalon and midbrain, and norepinephrine and serotonin variables in the hindbrain. Effects of diet were found only on serotonin and tryptophan in the subcortical regions. Significant multivariate correlation with the behavioral variables was demonstrated for the catecholamines but not for serotonin. The results suggest that these strain differences in behavior may be mediated by catecholamine systems.

Functional characterization of preganglionic neurons projecting in the lumbar splanchnic nerves: neurons regulating motility.

Lumbar preganglionic neurons, which projected in the lumbar splanchnic nerves and were probably involved in regulating motility of colon and pelvic organs (motility-regulating, MR neurons), were analyzed for their discharge patterns. The responses of the neurons to the following stimuli were tested: stimulation of arterial baro- and chemoreceptors and of afferents from the urinary bladder, colon, mucosal skin of the anus and perianal hairy skin. The following findings were made: a total of 131 preganglionic neurons were classified as MR neurons; these reacted to natural stimulation of at least one of the afferent inputs from the urinary bladder, colon and anal and perianal skin. The ongoing activity of these neurons did not correlate with the cardiac cycle or the cycle of the artificial ventilation. Most of them did not respond to an increase of blood pressure produced by i.v. injection of adrenaline or noradrenaline; some showed a weak depression or weak excitation which, in the time course, was untypical for visceral vasoconstrictor neurons. Stimulation of arterial chemoreceptors either did not influence MR neurons or produced only a secondary response owing to contraction of the urinary bladder. Ninety-seven preganglionic MR neurons could be subclassified: MR1 neurons were excited by distension and contraction of the urinary bladder and/or inhibited by distension and contraction of the colon (n = 61), a few were excited from both organs (n = 4); MR2 neurons were inhibited by distension and contraction of the urinary bladder and/or excited by distension and contraction of the colon (n = 32). Ninety-five out of 121 MR neurons (78.5%) were excited, 10 (8%) were inhibited and 16 (13%) not influenced by mechanical shearing stimuli applied to the mucosal skin of the anus. Most neurons which were excited by anal stimulation were not influenced by mechanical stimulation of the perianal (perigenital) skin. Twenty-eight per cent of the MR neurons (18 out of 64) were excited or inhibited upon stimulation of perianal skin. A few of these (7 out of 64 neurons, 11%) were involved in reflex responses which were different from those elicited from anal skin. At present no further consistent subclassification of MR1 and MR2 neurons appears possible on the basis of the excitatory and inhibitory anal and perianal reflexes. The results show that the population of visceral preganglionic neurons, which are probably involved in regulation of motility of colon and pelvic organs, is not homogeneous and probably consists of several subpopulations.

Functional characterization of preganglionic neurons projecting in the lumbar splanchnic nerves: vasoconstrictor neurons.

Lumbar preganglionic neurons, which project in the lumbar splanchnic nerves and which probably have a vasoconstrictor function (visceral vasoconstrictor, VVC neurons), were analyzed for their discharge patterns. The responses of these neurons to the following natural stimuli were tested: stimulation of arterial baroreceptors, arterial chemoreceptors and visceral afferents from the urinary bladder, the colon and the mucosal skin of the anus. Forty-nine preganglionic neurons were classified as VVC neurons. They showed the following characteristics: the ongoing activity of the VVC neurons exhibited pronounced cardiac rhythmicity and correlated with the cycle of the artificial ventilation. Stimulation of arterial baroreceptors, produced by increase of blood pressure or by increase of pressure in an isolated carotid blind sac, led to inhibition of activity in VVC neurons. Unloading of arterial baroreceptors, produced by decrease of blood pressure, led to an increase in VVC neuron activity. Stimulation of arterial chemoreceptors by bolus injections of CO2-enriched saline solution, close to a carotid glomus, led to a weak excitation of VVC neurons. Stimulation of arterial chemoreceptors by systemic hypoxia led to weak excitation and/or to depression of activity in VVC neurons. Stimulation of visceral afferents from urinary bladder and colon by isovolumetric contractions and distensions of the organs had no effect on most VVC neurons. Anal stimulation also did not induce reflexes in the majority of the VVC neurons. Some 14% of the VVC neurons (7 from 49) were excited by at least one of the visceral stimuli in the same manner as the motility-regulating (MR) neurons. This investigation shows that preganglionic neurons, probably involved in regulation of vascular resistance in colon and pelvic organs, are functionally a distinct population of neurons with some interesting functional overlap with the motility-regulating neurons.

Secondary functional properties of lumbar visceral preganglionic neurons.

Preganglionic visceral vasoconstrictor (VVC) neurons and motility-regulating (MR) neurons and other visceral preganglionic neurons, which project in the lumbar splanchnic nerves, were analyzed for their segmental distribution, the conduction velocity of their axons, ongoing activity and reflexes elicited by electrical stimulation of visceral afferents in white rami and of somatic afferents in spinal nerves. Identified preganglionic neurons and neurons without ongoing and reflex activity were distributed over segments L1-L5. VVC neurons were distributed over segments L1-L4 and MR neurons over segments L3-L5. VVC axons conducted at 2.8 +/- 2.5 m/s (mean +/- 1 S.D., n = 49), MR axons at 8.1 +/- 4.7 m/s (n = 131). The ongoing activity of VVC neurons was 1.6 +/- 0.7 imp/s (n = 46), that of MR neurons 0.8 +/- 0.7 imp/s (n = 91). There was no correlation between the conduction velocity of preganglionic axons and the rate of ongoing activity for VVC and MR neurons. (4) Electrical stimulation of visceral afferents in white rami and of somatic afferents in spinal nerves elicited short-latency (less than 50 ms) and long-latency (greater than 50 ms) reflexes in practically all VVC neurons, but preferentially short-latency reflexes in only 50 to 60% of the MR neurons. These results show that VVC and MR neurons are not only different in their reflex patterns, elicited by stimulation of visceral receptors and of arterial baro- and chemoreceptors, but also in the 4 properties analyzed in this paper.

Ubiquitin as a degradation signal.

For many short-lived eukaryotic proteins, conjugation to ubiquitin, yielding a multiubiquitin chain, is an obligatory pre-degradation step. The conjugated ubiquitin moieties function as a 'secondary' signal for degradation, in that their posttranslational coupling to a substrate protein is mediated by amino acid sequences of the substrate that act as a primary degradation signal. We report that the fusion protein ubiquitin--proline--beta-galactosidase (Ub-P-beta gal) is short-lived in the yeast Saccharomyces cerevisiae because its N-terminal ubiquitin moiety functions as an autonomous, primary degradation signal. This signal mediates the formation of a multiubiquitin chain linked to Lys48 of the N-terminal ubiquitin in Ub-P-beta gal. The degradation of Ub-P-beta gal is shown to require Ubc4, one of at least seven ubiquitin-conjugating enzymes in S.cerevisiae. Our findings provide the first direct evidence that a monoubiquitin moiety can function as an autonomous degradation signal. This generally applicable, cis-acting signal can be used to manipulate the in vivo half-lives of specific intracellular proteins.