Stokes's cradle: Newton's cradle with liquid coating.

Granular flows involving liquid-coated solids are ubiquitous in nature (pollen capture, avalanches) and industry (filtration, pharmaceutical mixing). In this Letter, three-body collisions between liquid-coated spheres are investigated experimentally using a "Stokes's cradle," which resembles the popular desktop toy Newton's cradle (NC). Surprisingly, previous work shows that every possible outcome was observed in the Stokes's cradle except the traditional NC outcome. Here, we experimentally achieve NC via guidance from a theory, which revealed that controlling the liquid-bridge volume connecting two target particles is the key in attaining the NC outcome. These three-body experiments also provide direct evidence that the fluid resistance upon rebound cannot be completely neglected due to presumed cavitation; this resistance also influences two-body systems yet cannot be isolated experimentally in such systems.

Purification of vacuoles from Neurospora crassa.

The Neurospora crassa vacuole, defined by its content of basic amino acids, polyphosphate, protease, phosphatases, and alpha-mannosidase, was purified to near homogeneity. The procedure depends upon homogenization of snail gut enzyme-digested cells in a buffer osmotically stabilized with 1 M sorbitol, differential centrifugation of the extract, and sucrose density gradient centrifugation of the organellar pellet. Isopycnic centrifugation of vacuoles in 2.25 M sorbitol-Metrizamide density gradients yielded a peak (density, 1.31 g/cm3) of vacuolar markers coincident with 32P-phospholipids, trichloroacetate-insoluble 14C, and trichloroacetate-soluble 14C. A trail of macromolecular markers in the lighter portions of the gradient reflected, at least in part, heterogeneity of the vacuoles. Almost no contamination by mitochondria or glyoxysomes was detected. Vacuoles were very heterogeneous in size as estimated by velocity sedimentation, but most were larger than mitochondria. Variations of the osmotic strength of the medium were found to alter the equilibrium density of vacuole preparations from 1.06 g/cm3 to over 1.3 g/cm3. This explains the great variation in density reported previously for the "vacuole," the "vesicle," and the "protease particle" of N. crassa, all of which appear to be the same entity.

Polyamine regulation of ornithine decarboxylase synthesis in Neurospora crassa.

Ornithine decarboxylase (ODC) of the fungus Neurospora crassa, encoded by the spe-1 gene, catalyzes an initial and rate-limiting step in polyamine biosynthesis and is highly regulated by polyamines. In N. crassa, polyamines repress the synthesis and increase the degradation of ODC protein. Changes in the rate of ODC synthesis correlate with similar changes in the abundance of spe-1 mRNA. We identify two sequence elements, one in each of the 5' and 3' regions of the spe-1 gene of N. crassa, required for this polyamine-mediated regulation. A 5' polyamine-responsive region (5' PRR) comprises DNA sequences both in the upstream untranscribed region and in the long 5' untranslated region (5'-UTR) of the gene. The 5' PRR is sufficient to confer polyamine regulation to a downstream, heterologous coding region. Use of the beta-tubulin promoter to drive the expression of various portions of the spe-1 transcribed region revealed a 3' polyamine-responsive region (3' PRR) downstream of the coding region. Neither changes in cellular polyamine status nor deletion of sequences in the 5'-UTR alters the half-life of spe-1 mRNA. Sequences in the spe-1 5'-UTR also impede the translation of a heterologous coding region, and polyamine starvation partially relieves this impediment. The results show that N. crassa uses a unique combination of polyamine-mediated transcriptional and translational control mechanisms to regulate ODC synthesis.

Nonsense mutations of the ornithine decarboxylase structural gene of Neurospora crassa.

Ornithine decarboxylase (ODC) (EC 4.1.1.17) is an early enzyme of polyamine synthesis, and its activity rises quickly at the onset of growth and differentiation in most eucaryotes. Some have speculated that the enzyme protein may have a role in the synthesis of rRNA in addition to its role in catalyzing the decarboxylation of ornithine (G. D. Kuehn and V. J. Atmar, Fed. Proc. 41:3078-3083, 1982; D. H. Russell, Proc. Natl. Acad. Sci. USA 80:1318-1321, 1983). To test this possibility, we sought mutational evidence for the indispensability of the ODC protein for normal growth of Neurospora crassa. We found three new, ODC-deficient mutants that lacked ODC protein. Among these and by reversion analysis of an earlier set of mutants, we found that two ODC-deficient mutants carried nonsense mutations in the ODC structural gene, spe-1. Allele LV10 imparted a complete deficiency for enzyme activity (less than 0.006% of normal) and had no detectable ODC antigen. Allele PE4 imparted a weak activity to cells (0.1% of derepressed spe+ cultures) and encoded a lower-molecular-weight ODC subunit (Mr = 43,000) in comparison to that of the wild-type strain (Mr = 53,000). Strains carrying either mutation, like other spe-1 mutants, grew at a normal rate in exponential culture if the medium was supplemented with spermidine, the main end product of the polyamine pathway in N. crassa. Unless an antigenically silent, N-terminal fragment with an indispensable role persists in the LV10-bearing mutant, we conclude that the ODC protein has no role in the vegetative growth of this organism other than the synthesis of polyamines. The data extend earlier evidence that spe-1 is the structural gene for ODC in N. crassa. The activity found in mutants bearing allele PE4 suggests that the amino acids nearest the carboxy terminus do not contribute to the active site of the enzyme.

Structural gene for ornithine decarboxylase in Neurospora crassa.

To define the structural gene for ornithine decarboxylase (ODC) in Neurospora crassa, we sought mutants with kinetically altered enzyme. Four mutants, PE4, PE7, PE69, and PE85, were isolated. They were able to grow slowly at 25 degrees C on minimal medium but required putrescine or spermidine supplementation for growth at 35 degrees C. The mutants did not complement with one another or with ODC-less spe-1 mutants isolated in earlier studies. In all of the mutants isolated to date, the mutations map at the spe-1 locus on linkage group V. Strains carrying mutations PE4, PE7, and PE85 displayed a small amount of residual ODC activity in extracts. None of them had a temperature-sensitive enzyme. The enzyme of the PE85 mutant had a 25-fold higher Km for ornithine (5mM) than did the enzyme of wild-type or the PE4 mutant (ca. 0.2 mM). The enzyme of this mutant was more stable to heat than was the wild-type enzyme. These characteristics were normal in the mutant carrying allele PE4. The mutant carrying PE85 was able to grow well at 25 degrees C and weakly at 35 degrees C with ornithine supplementation. This mutant and three ODC-less mutants isolated previously displayed a polypeptide corresponding to ODC in Western immunoblots with antibody raised to purified wild-type ODC. We conclude that spe-1 is the structural gene for the ODC.

Ornithine decarboxylase gene of Neurospora crassa: isolation, sequence, and polyamine-mediated regulation of its mRNA.

Ornithine decarboxylase (ODC), which initiates the biosynthesis of the polyamines putrescine, spermidine, and spermine, is encoded by the spe-1 gene of the fungus Neurospora crassa. This gene and its cDNA have been cloned and sequenced. The gene has a single 70-nucleotide intron in the coding sequence. The cDNA, comprising the entire coding region, recognizes a single 2.4-kb mRNA in Northern (RNA) blots. The mRNA transcript, defined by S1 mapping, has an extremely long, 535-base leader without strong secondary-structure features or an upstream reading frame. The translational start of the protein is ambiguous: a Met-Val-Met sequence precedes the Pro known to be the N terminus of the ODC polypeptide. The polypeptide encoded by the N. crassa spe-1 gene (484 amino acids) has 46% amino acid identity with that of Saccharomyces cerevisiae (466 amino acids) and 42% with that of mouse (461 amino acids). Alignment of the longer N. crassa sequence with S. cerevisiae and mouse sequences creates gaps in different sites in the S. cerevisiae and mouse sequences, suggesting that N. crassa ODC is closer to an ancestral form of the enzyme than that of either yeast or mouse ODC. N. crassa ODC, which turns over rapidly in vivo in the presence of polyamines, has two PEST sequences, found in most ODCs and other proteins with rapid turnover. In striking contrast to other eucaryotic organisms, the variation in the rate of ODC synthesis in response to polyamines in N. crassa is largely correlated with proportional changes in the abundance of ODC mRNA. Spermidine is the main effector of repression, while putrescine has a weaker effect. However, putrescine accumulation appears to increase the amount of active ODC that is made from a given amount of ODC mRNA, possibly by improving its translatability. Conversely, prolonged starvation for both putrescine and spermidine leads to the differentially impaired translation of ODC mRNA.

Deoxyhypusine/hypusine formation on a 21,000-dalton cellular protein in a Neurospora crassa mutant in vivo and in vitro.

Hypusine formation on an 18,000-dalton cellular protein is a unique spermidine-dependent, post-translational modification that appears to be ubiquitous in mammalian cells. To determine whether this modification also exists in lower eukaryotes, we examined possible labeling in vitro and in vivo of cellular protein(s) by [3H]spermidine in a mutant strain of Neurospora crassa (arge-12 ota aga) in which ornithine and polyamine synthesis could be nutritionally manipulated. Because of poor uptake of polyamines in this organism, [3H]ornithine, the immediate precursor of polyamines, was used for the in vivo labeling experiment. Both in vitro and in vivo labeling resulted in a specific labeling of a 21,000-dalton protein. Radioactive hypusine was recovered from radiolabeled 21,000-dalton protein following acid hydrolysis. The in vitro labeling of the 21,000-dalton protein was dramatically stimulated by NAD+ and NADP+, but not by FMN or FAD, suggesting that an NAD+/NADP(+)-dependent oxidative cleavage of spermidine is involved in deoxyhypusine formation. Isoelectric focusing/sodium dodecyl sulfate two-dimensional gel analysis revealed three isoforms of the in vitro labeled 21,000-dalton protein, with pI values ranging from 5.2 to 6.5. In contrast, the 21,000-dalton protein metabolically labeled in vivo gave only one spot with a pI value of approx. 3.5.

The mathematics of SELEX against complex targets.

We have developed a computer model for the simulation of simultaneous SELEX against multiple targets. The model assumes equilibrium behavior for the formation of binary ligand:target complexes, and that there is no ligand:ligand or target:target interaction. Target concentrations, ligand concentrations, and affinity distributions of the initial ligand pool for each individual target may be set by the user. We have used this program to gain an understanding of how the presence of multiple targets affects the selection process. In most cases, we find that SELEX is capable of generating different ligands for the different targets in a heterogeneous mixture, regardless of large variations in target concentrations and ligand:target affinities. A low relative partitioning efficiency (the efficiency with which ligands complexed with a target are separated from free ligands) for a target in a mixture gives a greatly reduced rate of selection of high-affinity ligands to that target. The ratio of each high-affinity ligand to its individual target within a pool of ligands selected for binding against a mixture of targets is approximately proportional to the concentration of the target multiplied by the ligand:target partitioning efficiency.

Genetics of arginine biosynthesis in Neurospora crassa.

A large number of arginine-requiring mutants of Neurospora was isolated, using a strain already partially impaired in an enzyme of the pathway. Among the mutants, all previously described loci, except one, were represented, and several new loci were defined and mapped. Four groups of mutants were of particular interest. First, the large group of arg-6 mutants, when tested for intragenic complementation, suggested a bifunctional gene, possibly controlling two steps in ornithine synthesis. This is consistent with the limited enzymic information about this locus. Second, the arg-13 locus was represented by 14 new mutants. All five tested were quite leaky, suggesting that the function controlled by this gene can be carried out to a limited extent spontaneously or by another gene product. Third, a new locus, arg-14, was defined. It controls a step in ornithine syntheses. It lies in a 1 to 2 map-unit interval between arg-2 and pyr-3 on LG IVR, as shown by mapping in relation to translocation breakpoints. Fourth, a second new locus whose mutants render the partial mutation in starting material auxotrophic was defined and mapped near the centromere of LG VIL. These new mutants are unable to derepress enzymes of the pathway and may qualify as regulatory mutants.

Neurospora mutants affecting polyamine-dependent processes and basic amino acid transport mutants resistant to the polyamine inhibitor, alpha-difluoromethylornithine.

Polyamines (spermidine and spermine) are required by living cells, but their functions are poorly understood. Mutants of Neurospora crassa with enhanced or diminished sensitivity to interference with polyamine synthesis, originally selected to study the regulation of the pathway, were found to have unexpected defects. A group of four non-allelic mutations, causing no interference with polyamine synthesis, each imparted spermidine auxotrophy to a genotype already partially impaired in spermidine synthesis. Strains carrying only the new mutations displayed unconditional delay or weakness at the onset of growth, but grew well thereafter and had a normal or overly active polyamine pathway. These mutants may have defects in vital macromolecular activities that are especially dependent upon the polyamines-activities that have not been identified with certainty in studies to date. Another group of mutants, selected as resistant to the polyamine inhibitor difluoromethylornithine (DFMO), had normal activity and regulation of ornithine decarboxylase, the target of the drug. All but one of thirty mutants were allelic, and were specifically deficient in the basic amino acid permease. This mechanism of DFMO resistance is unprecedented among the many DFMO-resistant cell types of other organisms and demonstrates that DFMO can be used for efficient genetic studies of this transport locus in N. crassa.

Large-scale oligoribonucleotide production.

Growing interest in RNAs for biomedical, agricultural, and other applications has necessitated the development of methods for producing oligoribonucleotides in milligram, or larger, quantities. Recent research on in vitro transcription has focused on optimizing solution conditions and the use of immobilized templates. In the alternative area of organic chemical synthesis, significant effort has been channeled into improving coupling efficiencies and producing modified RNA analogs which retain activity but have higher stability than unmodified RNAs.

Effect of abdominal transplanted mammary tumor on peritoneal fluid cytology in mice.

In this study C3H/HeJ female mice received an intraperitoneal injection of mammary tumor suspended in saline; After 14 days, the presence of mammary neoplasm was detected by a significant change in the cytodifferential counts of aspirated peritoneal fluid. Previous studies suggest that ovarian cancer can be detected in mice and in women by the characteristic changes produced in abdominal cellular fluid distribution.

Chronic occlusion of the monkey fallopian tube with silicone polymer.

Investigators have previously reported good retention, antifertility efficacy, and oviduct polymer compatibility in rabbit oviducts in studies of both short-term and chronic occlusion with high viscosity silicone polymer. The present study attempts to determine the chronic effects of the silicone polymer installed into the oviducts of 10 rhesus monkeys. Unlike former studies with rabbits, the polymer was installed into the fimbrial end of the monkey's oviducts because of the complexity of the uterotubal junction's musculature. Studies with light and scanning electron microscopy were conducted to examine tissue reaction to plug retention. Despite our major modification of the installation technique, we obtained a retention rate of 50%. In addition, there was no evidence of inflammation or tissue distortion, and the polymer plugs were not affected by menstrual bleeding.

Bactericidal action of nafcillin, vancomycin, and three cephalosporins against nafcillin-susceptible and nafcillin-resistant coagulase-negative staphylococci.

Coagulase-negative staphylococci (S. epidermidis, 43 strains; S. warneri, 16 strains; S. haemolyticus, five strains; and others, four strains) were tested by the agar dilution method for nafcillin susceptibility: 53 were susceptible with a minimal inhibitory concentration (MIC) of less than or equal to 2 micrograms/ml; four were of indeterminate susceptibility, MIC = 4-16 micrograms/ml; and 11 were resistant, MIC greater than or equal to 32 micrograms/ml. The bactericidal activities from 0 to 24 hr for nafcillin, vancomycin, cephalothin, cefazolin, and cefamandole, each at 16 micrograms/ml in broth, were determined for all the isolates. The data indicate that a nafcillin agar dilution susceptibility test result of resistance does not consistently predict lack of killing activity by the cephalosporins. It is likely that each cephalosporin would have to be tested against individual coagulase-negative staphylococci in order to determine a suitable therapeutic or prophylactic cephalosporin, if a cephalosporin were to be used. Vancomycin was bactericidal for all the nafcillin-resistant coagulase-negative organisms tested.

Modeling of repeated-batch transcription for production of RNA.

Enzymatic transcription for in vitro production of ribonucleic acids (RNA) is typically carried out in small batch reactors which suffer from low yields and high costs due to the formation of abortive RNA transcripts and the disposal of the DNA template and polymerase enzyme after a single use. This work considers repeated-batch transcription in which the DNA template molecules are immobilized on beads which are recovered and reused in multiple rounds. Some of the enzyme binds to the DNA template and is also recovered and reused. A model of this process is presented which employs equilibrium binding between the enzyme and template and which includes a first-order sequential deactivation of the enzyme. The model predicts the yields of RNA product and aborts for each round of repeated-batch transcription with no DNA addition after the first round and only partial enzyme replacement. The yield of RNA product per substrate (nucleoside triphosphates) generally decreases in subsequent rounds, whereas the yields of RNA product per enzyme and per template increase due to their reuse. Experimental data are presented which confirm the model and which show how the model parameters are obtained. A cost analysis shows that the cost of RNA production can be reduced by more than 50% for the system tested by employing an optimum number of rounds of repeated-batch transcription.

Long-term response to subtotal colectomy in colonic inertia.

The purpose of this study was to determine the long-term outcome of patients who had previously undergone subtotal colectomy for severe idiopathic constipation at the University of Florida between 1983 and 1987. In addition, we aimed to determine whether preoperative motility abnormalities of the upper gastrointestinal tract are more common among those patients who have significant postoperative complications after subtotal colectomy. We evaluated 13 patients who underwent subtotal colectomy for refractory constipation between 1983 and 1987 at the University of Florida. Preoperatively, all patients exhibited a pattern consistent with colonic inertia as demonstrated by means of radiopaque markers. Each patient was asked to quantitate the pain intensity and frequency of their bowel movements before and after surgery. In seven patients an ileosigmoid anastomosis was performed, whereas in six patients an ileorectal anastomosis was used. Abdominal pain decreased after subtotal colectomy. Patients with abnormal upper gastrointestinal motility preoperatively experienced greater postoperative pain than those with normal motility regardless of the type of anastomosis. In addition, the number of postoperative surgeries was similar in those patients with abnormal upper motility compared to those with normal motility. Overall, the total number of bowel movements per week increased from 0.5 +/- 0.03 preoperatively to 15 +/- 4.5 (P < 0.007) postoperatively. The results of our study suggest that patients with isolated colonic inertia have a better long-term outcome from subtotal colectomy than patients with additional upper gastrointestinal motility abnormalities associated with their colonic inertia.

RNA transcription from immobilized DNA templates.

We describe an RNA transcription protocol based on the multiple reuse of solid-phase synthetic DNA templates. The templates are assembled onto streptavidin-coated agarose beads via a single 5'-terminal biotin located on the noncoding template strand. Transcription occurs in an aqueous buffered suspension containing solid-phase DNA, dissolved enzyme (T7 RNA polymerase), and nucleoside triphosphate substrates (NTPs). A direct comparison of solution and solid-phase templates under standard transcription conditions reveals similar initial reaction rates and overall yields. Immobilized templates store stably for periods of several months and are easily recovered by mild centrifugation. We demonstrate the successive reuse of these templates throughout 15 rounds of transcription. The templates remained active, although an incremental decay in transcription was observed beyond five rounds. Template activity was partially restored by supplementing the support-bound oligonucleotide with fresh coding-strand DNA. These findings indicate that multiple reuse of template is a viable strategy for reducing the amount of DNA template required in RNA transcription.