The relationship between cell size and cell fate in Volvox carteri.

In Volvox carteri development, visibly asymmetric cleavage divisions set apart large embryonic cells that will become asexual reproductive cells (gonidia) from smaller cells that will produce terminally differentiated somatic cells. Three mechanisms have been proposed to explain how asymmetric division leads to cell specification in Volvox: (a) by a direct effect of cell size (or a property derived from it) on cell specification, (b) by segregation of a cytoplasmic factor resembling germ plasm into large cells, and (c) by a combined effect of differences in cytoplasmic quality and cytoplasmic quantity. In this study a variety of V. carteri embryos with genetically and experimentally altered patterns of development were examined in an attempt to distinguish among these hypotheses. No evidence was found for regionally specialized cytoplasm that is essential for gonidial specification. In all cases studied, cells with a diameter > approximately 8 microns at the end of cleavage--no matter where or how these cells had been produced in the embryo--developed as gonidia. Instructive observations in this regard were obtained by three different experimental interventions. (a) When heat shock was used to interrupt cleavage prematurely, so that presumptive somatic cells were left much larger than they normally would be at the end of cleavage, most cells differentiated as gonidia. This result was obtained both with wild-type embryos that had already divided asymmetrically (and should have segregated any cytoplasmic determinants involved in cell specification) and with embryos of a mutant that normally produces only somatic cells. (b) When individual wild-type blastomeres were isolated at the 16-cell stage, both the anterior blastomeres that normally produce two gonidia each and the posterior blastomeres that normally produce no gonidia underwent modified cleavage patterns and each produced an average of one large cell that developed as a gonidium. (c) When large cells were created microsurgically in a region of the embryo that normally makes only somatic cells, these large cells became gonidia. These data argue strongly for a central role of cell size in germ/soma specification in Volvox carteri, but leave open the question of how differences in cell size are actually transduced into differences in gene expression.

Heat shock elicits production of sexual inducer in Volvox.

In the green alga Volvox carteri, heat shock had an unusual and adaptive effect mediated by induced production of a well-defined effector molecule. Females of this species normally reproduce asexually in the absence of a potent sexual inducer produced by mature sexual males, but they generated egg-bearing sexual daughters after a brief exposure to elevated temperatures. This response involved an "autoinduction" of sexuality, in which heat-shocked somatic cells made and released the sexual inducer, which then redirected development of the reproductive cells. Males, including a sterile mutant incapable of producing inducer in the usual manner, also produced the inducer in response to heat shock. The phenomenon probably is of significance in the wild, where Volvox reproduces asexually in temporary ponds in spring but becomes sexual and produces dormant, overwintering zygotes before the ponds dry up in the summer heat.

Temporal decline in defibrillation thresholds with an active pectoral lead system.

OBJECTIVES: The objective of this study was to characterize temporal changes in defibrillation thresholds (DFTs) after implantation with an active pectoral, dual-coil transvenous lead system. BACKGROUND: Ventricular DFTs rise over time when monophasic waveforms are used with non-thoracotomy lead systems. This effect is attenuated when biphasic waveforms are used with transvenous lead systems; however, significant increases in DFT still occur in a minority of patients. The long-term stability of DFTs with contemporary active pectoral lead systems is unknown. METHODS: This study was a prospective assessment of temporal changes in DFT using a uniform testing algorithm, shock polarity and dual-coil active pectoral lead system. Thresholds were measured at implantation, before discharge and at long-term follow-up (70 +/- 40 weeks) in 50 patients. RESULTS: The DFTs were 9.2 +/- 5.4 J at implantation, 8.3 +/- 5.8 J before discharge and 6.9 +/- 3.6 J at long-term follow-up (p < 0.01 by analysis of variance; p < 0.05 for long-term follow-up vs. at implantation or before discharge). The effect was most marked in a prespecified subgroup with high implant DFTs (> or =15 J). No patient developed an inadequate safety margin (< 9 J) during follow-up. CONCLUSIONS: The DFTs declined significantly after implantation with an active pectoral, dual-coil transvenous lead system, and no clinically significant increases in DFT were observed. Therefore, routine defibrillation testing may not be required during the first two years after implantation with this lead system, in the absence of a change in the cardiac substrate or treatment with antiarrhythmic drugs.

Chronic rise in monophasic defibrillation thresholds with a transvenous lead system.

This study was a prospective evaluation of chronic changes of defibrillation thresholds in 31 clinically stable patients with a single transvenous lead, optimal shock polarity, and uniform testing protocol. At a mean follow-up of 273 +/- 146 days, defibrillation thresholds increased 26%, from 13.2 +/- 5.6 J to 17.1 +/- 6:0 J (p < 0.001), and shock impedance increased from 46.2 +/- 7.0 omega to 51.2 +/- 6.2 omega (p < 0.001).

Reversible decerebrate posturing after profound and prolonged hypoglycemia.

Decerebrate rigidity is one of several reversible neurological abnormalities which have been observed in the setting of metabolic coma. We present the case of a patient who recovered fully from prolonged decerebrate rigidity associated with hypoglycemic coma. This case emphasizes the possibility of recovery from severe, prolonged hypoglycemia.

Current approaches to evaluation and management of patients with ventricular arrhythmias.

The clinical manifestations of ventricular arrhythmias encompass a broad spectrum, from complete absence of symptoms to sudden death. Although our understanding of the pathophysiology and natural history of these arrhythmias has advanced significantly over the past decade, large gaps in our knowledge remain, especially in patients with heart failure not due to coronary artery disease. We have learned much about the appropriate roles of antiarrhythmic drugs and implantable defibrillators in the prevention of sudden death. Studies performed over the past decade have made clear that the primary treatment for patients at high risk for life-threatening ventricular arrhythmias should be the implantable defibrillator. However, specific syndromes causing ventricular tachyarrhythmias are being recognized, and care must be individualized. Although hospital mortality from acute myocardial infarction has decreased as a result of newer therapies, sudden death after hospital discharge remains an important problem, causing at least 30% of post-infarction deaths, even in patients who have received thrombolytic therapy. Two independent studies have confirmed that patients with asymptomatic non-sustained ventricular tachycardia in the presence of left ventricular ejection fraction < .40 after myocardial infarction who have sustained ventricular tachycardia inducible by electrophysiologic study are at significant risk for sudden death. This risk is significantly reduced by ICD, but not pharmacologic, antiarrhythmic therapy. Our major challenge at this time is not how best to treat high risk patients, but how best to identify them prior to events. Finally, physicians should be aware that many symptomatic ventricular tachycardias are now curable at low risk, using catheters to deliver radiofrequency energy.

Carrier-mediated Uptake of Arginine and Urea by Volvox carteri f. nagariensis.

Volvox carteri f. nagariensis takes up arginine via a high affinity, highly specific carrier, whereas carriers for neutral and acidic amino acids cannot be detected (even in nitrogen-starved cultures). Exogenous arginine is accumulated against a steep concentration gradient and is incorporated into protein with high efficiency, but it is not catabolized to any significant extent and will not serve as a nitrogen source adequate to support growth. Urea is also taken up by a saturable carrier, but several lines of evidence indicate that the arginine and urea carriers are distinct and different. Preexposure to arginine suppresses arginine uptake while stimulating urea uptake. The K(i) values observed for reciprocal, competitive inhibition of uptake by arginine and urea are orders of magnitude different from the respective K(m) values for uptake. The two uptake systems show entirely different patterns of sensitivity to inhibition by structural analogs. Finally, the V(max) values for arginine and urea uptake fluctuate independently (but in a regular pattern) during the asexual life cycle. The fluctuations of urea uptake activity are of considerable magnitude and appear to be linked to key phases of the developmental program.

Carrier-mediated Uptake of Arginine and Urea by Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii possesses a high affinity, highly specific carrier involved in uptake of exogenous arginine. Carrier-mediated uptake of other amino acids cannot be detected, even in cultures maintained on amino acids as a nitrogen source or starved for nitrogen. This fact may contribute to the difficulty of isolating strains auxotrophic for amino acids other than arginine; conventional selection media may not supply adequate quantities of amino acids to permit growth of auxotrophs. A urea carrier is also present in C. reinhardtii but is readily distinguished from the arginine carrier on the basis of kinetic properties and sensitivity to a range of structural analogs. Ammonia appears to play a major role in regulating (depressing) activity of the arginine uptake system. Activity of the urea uptake system is elevated in nitrogen-starved cultures and elevated even further in the presence of urea or arginine. Extensive, independent fluctuations in the two uptake systems observed in semisynchronous cultures suggest that both are subject to modulation by a complex set of interacting endogenous and exogenous factors.

Protein synthetic patterns during the asexual life cycle of Volvox carteri.

The polypeptide labeling patterns of somatic cells, gonidia (asexual reproductive cells), embryos, and juvenile spheroids of Volvox carteri cultures synchronized by a light/dark cycle were studied as a function of developmental stage and incubation condition. Specimens were exposed to 35SO=4 for 1-hr periods at selected intervals throughout the asexual life cycle; proteins were then extracted and analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fluorography. Although sulfation accounts for more than half the 35S incorporated, the conditions of extraction and electrophoresis employed resulted in exclusion of most sulfated products and inclusion of nearly all products bearing incorporated sulfur amino acids in the electrophoretic analysis. Hence SDS-PAGE profiles reflect relative rates of synthesis of major polypeptides. The first phase of these studies involved examination of stage-specific differences in protein synthetic patterns. Because a single developmental stage exhibits different protein synthetic patterns in light and darkness, detailed developmental comparisons were made only on organisms or cells exposed to label in the light. They yielded the following results: Shortly after the completion of embryogenesis (while all cells are still linked by numerous cytoplasmic bridges) presumptive somatic cells and gonidia exhibit a nearly identical pattern of labeling of the major polypeptides. In just a few hours, however, as cytoplasmic bridges begin to break down, the synthetic patterns of the two cell types begin to diverge; with passing time this divergence becomes progressively greater. By the time gonidia are mature, the patterns of labeling of major polypeptides by somatic cells and gonidia exhibit far more differences than similarities. Embryos derived from these mature gonidia then exhibit numerous, reproducible, stage-specific changes in polypeptide labeling throughout embryogenesis. However, two glycoproteins that previous authors implicated in the control of the differentiative cleavage division are here shown to be labeled in the parental somatic cells, not in the embryos as was previously supposed; hence a central role for them in embryonic development seems highly unlikely. In the second phase of this study the effects of light on protein synthetic patterns of organisms at selected developmental stages were analyzed. At all stages marked, rapid, reversible changes in the pattern of labeling of major polypeptides occur when cultures are transferred from light to dark or vice versa, but these changes are most marked in juvenile spheroids at the end of the dark period during which they had completed their embryogenesis. Some, but by no means all, of the changes induced by light can be attributed to stimulated synthesis of chloroplast proteins, on both chloroplast and cytosol ribosomes. The proteins made at the beginning of one light period are not identical to those made at the end of the preceding light period...

Translational regulation of protein synthesis, in response to light, at a critical stage of Volvox development.

In Volvox cultures synchronized by a light-dark cycle, juveniles containing presumptive somatic and reproductive cells are produced during the dark, but their cells do not differentiate until after the lights come on. The pattern of protein synthesis changes rapidly after the lights come on. Action spectra and effects of photosynthesis inhibitors indicate that this protein synthetic change is not simply a consequence of renewed flow of energy from illuminated chloroplasts. Actinomycin, at a level adequate to block the response to heat shock, has virtually no effect on the response of the same cells to light; furthermore, RNAs isolated from unilluminated and illuminated juveniles yield indistinguishable in vitro translation products. We conclude, therefore, that this effect of light is exerted almost exclusively at the translational level, generating one of the most striking examples of translational regulation yet described.

Deployment of a previously embolized, unexpanded, and disarticulated Palmaz-Schatz stent.

Stent embolization is a rare but acknowledged complication of placement of disarticulated (half) Palmaz-Schatz stents. We report a case in which we diagnosed a previously unrecognized, embolized, undeployed half-stent in the distal LAD, causing slow flow, and then deployed the stent where it lay, resulting in improved flow. The literature on treatment of coronary stent embolization and on cutting and preparing half-stents for deployment is discussed.

Molecular phylogeny of the volvocine flagellates.

Phylogenetic studies of approximately 2,000 bases of sequence from the large and small nuclear-encoded ribosomal RNAs are used to investigate the origins of the genus Volvox. The colonial and multicellular genera currently placed in the family Volvocaceae form a monophyletic group that is significantly closer phylogenetically to Chlamydomonas reinhardtii than it is to the other unicellular green flagellates that were tested, including Chlamydomonas eugametos, Chlorella pyrenoidosa, and Haematococcus lacustris. Statistical analysis of 251 phylogenetically informative nucleotide positions rejects the "volvocine lineage" hypothesis, which postulates a monophyletic evolutionary progression from unicellular organisms (such as Chlamydomonas), through colonial organisms (e.g., Gonium, Pandorina, Eudorina, and Pleodorina) demonstrating increasing size, cell number, and tendency toward cellular differentiation, to multicellular organisms having fully differentiated somatic and reproductive cells (in the genus Volvox). The genus Volvox appears not to be monophyletic. Volvox capensis falls outside a lineage containing other representatives of Volvox (V. aureus, V. carteri, and V. obversus), and both of these Volvox lineages are more closely related to certain colonial genera than they are to each other. This implies either a diphyletic origin of Volvox from different colonial volvocacean ancestors, a phylogenetic derivation of some of the colonial genera from a multicellular (i.e., Volvox) ancestor, or both. Considered together with previously published observations, these results suggest that the different levels of organizational and developmental complexity found in the Volvocaceae represent alternative stable states, among which evolutionary transitions have occurred several times during the phylogenetic history of this group.

Nuclear transformation of Volvox carteri.

Stable nuclear transformation of Volvox carteri was achieved using the cloned V. carteri nitA+ gene (which encodes nitrate reductase) to complement a nitA- mutation. Following bombardment of mutant cells with plasmid-coated gold particles, putative transformants able to utilize nitrate as a nitrogen source were recovered with an efficiency of approximately 2.5 x 10(5). DNA analysis indicated that the plasmid integrated into the genome, often in multiple copies, at sites other than the nitA locus. Cotransformants were recovered with a frequency of 40-80% when cells were cobombarded with a selected and an unselected marker. Thus, V. carteri becomes one of the simplest multicellular organisms that is accessible to detailed molecular studies of genes regulating cellular differentiation and morphogenesis.

Patterns of organellar and nuclear inheritance among progeny of two geographically isolated strains of Volvox carteri.

Strains of Volvox carteri forma nagariensis derived from Japanese and Indian isolates ("J" and "I" strains, respectively) exhibited length differences (RFLPs) for approximately 90% of the restriction fragments detected by hybridization with a variety of unique-sequence, small-gene-family and repetitive-element probes, including heterologous probes of chloroplast and mitochondrial origin. Extensive post-zygotic mortality was observed among the zygotes produced by crossing J and I strains, suggesting some form of genetic incompatability between them. Most of the viable progeny exhibited recombinant patterns of nuclear inheritance and maternal inheritance of mitochondrial and chloroplast markers. However, many progeny exhibited exclusively uniparental (usually maternal, but in one case paternal) inheritance of both nuclear and organellar markers. Some of these non-recombinant individuals may be derived from "parthenospores" (dormant asexual cells resembling zygospores). Others may be a result of "pseudogamy," in which one of the parental pronuclei is excluded from the zygote, followed by selective exclusion of both the mitochondrial and the chloroplast genomes derived from that same parent. When segregation patterns for 44 nuclear markers were analyzed in 90 recombinant progeny, statistically significant, locus-specific deviations from expected Mendelian transmission ratios were observed for a sizeable fraction of all markers in both reciprocal crosses: some markers were preferentially transmitted by the J strain, while others were preferentially transmitted by the I strain. It is speculated that these transmission distortions may be related to the regions of inter-isolate genetic incompatibility, and may complicate the use of J x I crosses to establish a RFLP-based linkage map for the species.

regA, a Volvox gene that plays a central role in germ-soma differentiation, encodes a novel regulatory protein.

Volvox has two cell types: mortal somatic cells and immortal germ cells. Here we describe the transposon-tagging, cloning and characterization of regA, which plays a central role as a master regulatory gene in Volvox germ-soma differentiation by suppressing reproductive activities in somatic cells. The 12.5 kb regA transcription unit generates a 6,725 nucleotide mRNA that appears at the beginning of somatic cell differentiation, and that encodes a 111 kDa RegA protein that localizes to the nucleus, and has an unusual abundance of alanine, glutamine and proline. This is a compositional feature shared by functional domains of many 'active' repressors. These findings are consistent with the hypothesis that RegA acts in somatic cells to repress transcription of genes required for growth and reproduction, including 13 genes whose products are required for chloroplast biogenesis.