Genetic Modification of Grapevine Embryogenic Cultures.

Precision breeding is an approach to grapevine genetic improvement that transfers only specific traits among sexually compatible species via the relatively stable mitotic cell division pathway in order to avoid the significant disruption imposed upon conventional breeding by meiosis. Factors enabling precision breeding include the availability of the Vitis genome sequence combined with highly optimized gene insertion and plant regeneration protocols. A protocol for the production of grapevine embryogenic cultures and their genetic transformation is described. Embryogenic cultures are produced from either leaf or floral explants. Somatic embryos at the cotyledonary stage of development are used for Agrobacterium-mediated transformation. Following co-cultivation with Agrobacterium containing the genes of interest, modified embryos are selected on the basis of anthocyanin pigmentation and antibiotic resistance. Somatic embryos are then germinated to produce modified plants that are hardened and transferred to a greenhouse. The presence of the genes of interest is confirmed by PCR.

Intercomparison of methods of coupling between convection and large-scale circulation: 2. Comparison over nonuniform surface conditions.

As part of an international intercomparison project, the weak temperature gradient (WTG) and damped gravity wave (DGW) methods are used to parameterize large-scale dynamics in a set of cloud-resolving models (CRMs) and single column models (SCMs). The WTG or DGW method is implemented using a configuration that couples a model to a reference state defined with profiles obtained from the same model in radiative-convective equilibrium. We investigated the sensitivity of each model to changes in SST, given a fixed reference state. We performed a systematic comparison of the WTG and DGW methods in different models, and a systematic comparison of the behavior of those models using the WTG method and the DGW method. The sensitivity to the SST depends on both the large-scale parameterization method and the choice of the cloud model. In general, SCMs display a wider range of behaviors than CRMs. All CRMs using either the WTG or DGW method show an increase of precipitation with SST, while SCMs show sensitivities which are not always monotonic. CRMs using either the WTG or DGW method show a similar relationship between mean precipitation rate and column-relative humidity, while SCMs exhibit a much wider range of behaviors. DGW simulations produce large-scale velocity profiles which are smoother and less top-heavy compared to those produced by the WTG simulations. These large-scale parameterization methods provide a useful tool to identify the impact of parameterization differences on model behavior in the presence of two-way feedback between convection and the large-scale circulation.

Intercomparison of methods of coupling between convection and large-scale circulation: 1. Comparison over uniform surface conditions.

As part of an international intercomparison project, a set of single-column models (SCMs) and cloud-resolving models (CRMs) are run under the weak-temperature gradient (WTG) method and the damped gravity wave (DGW) method. For each model, the implementation of the WTG or DGW method involves a simulated column which is coupled to a reference state defined with profiles obtained from the same model in radiative-convective equilibrium. The simulated column has the same surface conditions as the reference state and is initialized with profiles from the reference state. We performed systematic comparison of the behavior of different models under a consistent implementation of the WTG method and the DGW method and systematic comparison of the WTG and DGW methods in models with different physics and numerics. CRMs and SCMs produce a variety of behaviors under both WTG and DGW methods. Some of the models reproduce the reference state while others sustain a large-scale circulation which results in either substantially lower or higher precipitation compared to the value of the reference state. CRMs show a fairly linear relationship between precipitation and circulation strength. SCMs display a wider range of behaviors than CRMs. Some SCMs under the WTG method produce zero precipitation. Within an individual SCM, a DGW simulation and a corresponding WTG simulation can produce different signed circulation. When initialized with a dry troposphere, DGW simulations always result in a precipitating equilibrium state. The greatest sensitivities to the initial moisture conditions occur for multiple stable equilibria in some WTG simulations, corresponding to either a dry equilibrium state when initialized as dry or a precipitating equilibrium state when initialized as moist. Multiple equilibria are seen in more WTG simulations for higher SST. In some models, the existence of multiple equilibria is sensitive to some parameters in the WTG calculations.

Structure and evolution of supernumerary chromosomes in the Pacific giant salamander, Dicamptodon tenebrosus.

We examined the genetic make-up and plausible origins of the supernumerary (B) chromosomes of the Pacific giant salamander, Dicamptodon tenebrosus, from the Pacific Northwest of North America. These salamanders have variable numbers of B chromosomes, from 0 to 10 per individual. Salamanders from the most southerly and northerly regions of the species' range have lower average numbers of B chromosomes than salamanders in the middle of the range. To assess how the supernumerary chromosomes originated in D. tenebrosus, B chromosome DNA was isolated by microdissection and amplified by degenerate oligonucleotide-primed PCR. The B chromosome DNA hybridized similarly to genomic DNA from individuals of D. tenebrosus and the related species D. copei and D. ensatus, thus demonstrating that the supernumerary chromosomes were derived from the normal chromosome complement. Unique hybridization bands in both D. copei and D. tenebrosus suggest that the shared sequences have evolved independently.

Comparative risk assessment: an international comparison of methodologies and results.

Comparative risk assessment (CRA) is a systematic procedure for evaluating the environmental problems affecting a geographic area. This paper looks beyond the U.S. border and examines the experience with CRAs conducted in various developing countries and economies in transition, including Bangkok, Thailand, Cairo, Egypt and Quito, Ecuador, as well as other locations in Eastern Europe, Asia and Central and South America. A recent pilot CRA conducted in Taiwan is also considered. Comparisons are made of both the methodologies and the results across the relatively diverse international literature. The most robust finding is that conventional air pollutants (e.g., particulate matter and lead) consistently rank as high health risks across all of the CRAs examined. Given the varied nature of the settings studied in the CRAs, including level of economic development, urban-rural differences, and climate, this finding is particularly significant. Problems involving drinking water are also ranked as a high or medium health risk in almost all the countries studied. This is consistent with the results of analyses conducted by the World Bank suggesting contamination, limited coverage and erratic service by water supply systems. Beyond the major air pollutants and drinking water, the CRA results diverge significantly across countries. A number of problems involving toxic chemicals, e. g., hazardous air pollutants, rank as high health risks in the US but do not appear as consistent areas of concerns in the other countries studied. This likely reflects the so-called "risk transition" - the shift from sanitation and infection disease problems to those involving industry, vehicles and toxic substances - that often occurs with economic development. It may also reflect the greater information about sources of toxic pollutants in the U.S. For other problems, there are important differences across the developing countries and economies in transition. For example, hazardous and (industrial) non-hazardous waste issues ranked as medium or low health risks in all the countries studied, except for Taiwan where unmanaged toxic waste sites were considered to pose high risks. While the generally low ranking is consistent with the notion that few people are directly exposed to hazardous and (industrial) non-hazardous waste, it is not entirely surprising that views might be different in Taiwan, where space is so limited and population density is so high. We suggest that the wide range of findings likely reflect genuine differences among the countries studied. However, we cannot entirely rule out the possibility that some of the observed similarities (and differences) arise from the (relatively) common methodologies employed.

Morphological clues from multilegged frogs: are retinoids to blame?

Morphological analysis was performed on multilegged deformed frogs representing five species from 12 different localities in California, Oregon, Arizona, and New York. The pattern of duplicated limbs was consistent with mechanical perturbation by trematode infestation but not with the effects of retinoids.

Alternative therapy for elderly patients with breast cancer.

Breast cancer treatment has undergone significant changes in concept, concurrent with alterations in our understanding of cancer biology and natural history. Within the last 10 years, oncologists have brought into question the traditional Halstedian concepts of the natural history of breast cancer and its appropriate management. The goal of treatment, once a primary cancer is detected in the breast, is to prevent metastasis and subsequent death of the patient. One hundred forty-two female patients over the age of 65 with histologically confirmed breast cancer were treated at Lankenau Hospital from 1982 to 1990. We treated 32 women over the age of 65 with quadrantectomy and tamoxifen as the sole form of therapy. No radiation, standard chemotherapy, nor axillary dissection was utilized. A cohort of 110 women of similar age, treated for breast cancer with "standard therapy" (total mastectomy or "segmental resection" and radiation with axillary nodal dissection) during the same time period, were also analyzed retrospectively. All segmental resections were followed by standard radiation doses to the ipsilateral breast and draining nodal basins with a local boost. Twenty-nine of 32 patients in the quad + tam group were available for follow-up 1 to 8 years following treatment (mean 52 months). The cumulative overall survival was 67 per cent and disease-free survival 92 per cent. No patient developed local recurrence. Follow-up analysis of the 110 women treated in "standard fashion" was complete in 88 patients 1 to 8 years post-treatment (mean 56 months). Cumulative overall survival was 82 per cent and disease-free survival 83 per cent. Local recurrence was noted in five per cent.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute acalculous cholecystitis following open heart surgery.

We undertook a retrospective study designed to ascertain the frequency of acute acalculous cholecystitis (AAC) following open heart surgery. In the study period 1982-1990, 22 of 6393 patients following open heart surgery were recognized to have developed AAC, an incidence of 0.34%. The majority of patients (16/22) presented within the first postoperative week. Vague right upper quadrant physical findings, nonspecific changes in the liver function chemistries and unexplained sepsis frequently led to radiologic evaluations. Ultrasonography was the most valuable radiologic study, with a diagnosis sensitivity of 82%. Technetium cholescintography can serve as a useful adjunct when interpreted in the context of other clinical findings. Cholecystectomy was performed in 20 patients and cholecystostomy in two. Nine (41%) patients had gangrenous gallbladders with frank perforation in two. A specific preoperative diagnosis was made in 19 patients (86%). Fifteen patients survived for a mortality rate of 32%. In 12 of 15 survivors (80%), the diagnosis of AAC was established and laparotomy performed within 48 hours of first clinical suspicion. Gangrene and perforation were seen in 87% of patients in whom surgery was delayed. AAC is a life-threatening condition especially in critically ill patients. Experience suggests that early diagnosis and operative intervention are the key elements of treatment. Delay of operative management on the grounds of recent cardiac surgery is not justified.

Cutaneous angiosarcoma of the breast after segmental mastectomy and radiation therapy.

Angiosarcoma of the breast is an uncommon entity, and the development of cutaneous angiosarcoma of the breast after segmental mastectomy and irradiation therapy is even less common. We report a case of cutaneous angiosarcoma that developed 4 1/2 years after segmental mastectomy (lumpectomy) with axillary dissection and irradiation therapy for infiltrating ductal carcinoma of the breast.

Diagnostic laparoscopy.

Laparoscopy is a unique procedure which can be used in diagnostic or therapeutic situations involving intra-abdominal pathology. Until recently, open laparotomy has been the preferred method for such evaluations. The following cases indicate the increased sensitivity, specificity, safety, and cost effectiveness of laparoscopy in diagnostic situations.

Effects of localized application of transforming growth factor beta 1 on developing chick limbs.

The effects of exogenous transforming growth factor beta (TGF-beta) on chick limb development in vivo were studied by implanting carriers of TGF-beta 1 into developing wing buds. Agarose beads were soaked in solutions containing TGF-beta 1 and implanted into wing buds at stages 18 to 27. Localized application of TGF-beta 1 to distal regions of the wing bud caused specific skeletal elements in the limb to be reduced or absent. The particular proximal-distal limb element affected depended on the stage at which the bead was implanted. Position of the bead in the anterior-posterior axis also influenced the pattern of affected structures. Experiments in which TGF-beta 1 beads were implanted and then removed at 24- and 48-hr intervals indicate that there are specific periods during which a skeletal element appears to be sensitive to the effects of exogenous TGF-beta 1. In a few cases, beads placed in proximal positions in later staged limbs resulted in formation of ectopic cartilage near the bead. These results suggest that exposure to exogenous TGF-beta 1 in vivo influences the development of skeletal elements in the chick limb in a stage- and position-dependent manner.

Explanation for naturally occurring supernumerary limbs in amphibians.

The occasional occurrence of high frequencies of limb abnormalities, including extra limbs, in natural populations of amphibians has long been a puzzle. In this paper we report the discovery of a population in which such limb abnormalities appear to be caused by a parasitic flatworm (trematode) that uses amphibians as intermediate hosts. The cercarial larval stage of the trematode attacks amphibians, penetrating the skin to form cysts (metacercariae). The cysts are preferentially localized in the cloacal region, including the developing hind limb regions in larvae of both frogs (Hyla regilla) and salamanders (Ambystoma macrodactylum). A wide range of limb abnormalities are seen, including duplicated limb structures ranging from extra digits to several extra whole limbs. We hypothesize that these limb abnormalities result from localized regulatory responses of developing and regenerating limb tissues to mechanical disruption caused by the trematode cysts. We have tested this idea by implanting inert resin beads into developing limb buds of frogs and salamanders. Since this treatment can cause supernumerary limb structures, our hypothesis is sufficient to explain the naturally occurring extra limbs.

Compatible limb patterning mechanisms in urodeles and anurans.

We have experimentally tested the similarity of limb pattern-forming mechanisms in urodeles and anurans. To determine whether the mechanisms of limb outgrowth are equivalent, we compared the results of two kinds of reciprocal limb bud grafts between Xenopus and axolotls: contralateral grafts to confront anterior and posterior positions of graft and host, and ipsilateral grafts to align equivalent circumferential positions. Axolotl limb buds grafted to Xenopus hosts are immunologically rejected at a relatively early stage. Prior to rejection, however, experimental (but not control) grafts form supernumerary digits. Xenopus limb buds grafted to axolotl hosts are not rejected within the time frame of the experiment and therefore can be used to test the ability of frog cells to elicit responses from axolotl tissue that are similar to those that are elicited by axolotl tissue itself. When Xenopus buds were grafted to axolotl limb stumps so as to align circumferential positions, the majority of limbs did not form any supernumerary digits. However, in experimental grafts, where anterior and posterior of host and graft were misaligned, supernumerary digits formed at positional discontinuities. These results suggest that Xenopus/axolotl cell interactions result in responses that are similar to axolotl/axolotl cell interactions. Furthermore, axolotl and Xenopus cells can cooperate to build recognizable skeletal elements, despite large differences in cell size and growth rate between the two species. We infer from these results that urodeles and anurans share the same limb pattern-forming mechanisms, including compatible positional signals that allow appropriate localized cellular interactions between the two species. Our results suggest an approach for understanding homology of the tetrapod limb based on experimental cellular interactions.

Cytology, embryology, and evolution of the developmental arrest syndrome in newts of the genus Triturus (Caudata: Salamandridae).

We have examined embryonic development in three species (T. carnifex, T. cristatus, and T. marmoratus) of European newts of the genus Triturus (subgenus Neotriton) in which developmental arrest occurs in embryos that are homomorphic for a chromosomal heteromorphism involving chromosome 1 (Horner and Macgregor: J. Herpetol., 19:261-270, 1985). Embryonic arrest occurred during tailbud stages in all three species, but at a slightly earlier stage in T. marmoratus. Two phenotypes were identified among the arrested embryos. One of these is indistinguishable in embryonic morphology from normal embryos at all stages up to the time of arrest, but the other is characterized by a protruding yolk plug, which persists from the late gastrula/early neurula stage to the tailbud arrest stage and apparently interferes with normal morphogenesis. Evidence is presented that the two arrested phenotypes, which occur in approximately equal numbers, represent embryos that carry the two alternative homomorphic chromosome pairs of chromosome 1 heteromorphism. We conclude that developmental arrest reflects a balanced lethal heterozygosity probably resulting from an unequal exchange of genic material between the homologues of chromosome 1 which occurred in a common ancestor of the Neotriton species.

Evidence that regenerative ability is an intrinsic property of limb cells in Xenopus.

Xenopus laevis exhibits an ontogenetic decline in the ability to regenerate its limbs: Young tadpoles can completely regenerate an amputated limb, whereas post metamorphic froglets regenerate at most a cartilagenous "spike." We have tested the regenerative competence of normally regenerating limb buds of stage 52-53 Xenopus tadpoles grafted onto limb stumps of postmetamorphic froglets. The limb buds become vascularized and innervated by the host and, when amputated, regenerate limbs with normal or slightly less than normal numbers of tadpole hindlimb digits. Reciprocal grafts of froglet forelimb blastemas onto tadpole hindlimb stumps resulted in either autonomous development of tadpole hindlimb structures and/or formation of a cartilaginous spike typical of froglet forelimb regeneration. Our results suggest that the Xenopus froglet host environment is completely permissive for regeneration and that the ability to regenerate a complete limb pattern is an intrinsic property of young tadpole limb cells, a property that is lost during ontogenesis.

The biological significance of variation in satellite DNA and heterochromatin in newts of the genus Triturus: an evolutionary perspective.

The functional and evolutionary significance of highly repetitive, simple sequence (satellite) DNA is analysed by examining available information on the patterns of variation of heterochromatin and cloned satellites among newts (family Salamandridae), and particularly species of the European genus Triturus. This information is used to develop a model linking evolutionary changes in satellite DNAs and chromosome structure. In this model, satellites accumulate initially in large tandem blocks around centromeres of some or all of the chromosomes, mainly by repeated chromosomal exchanges in these regions. Centromeric blocks later become broken up and dispersed by small, random chromosome rearrangements in these regions. They are dispersed first to pericentric locations and then gradually more distally into the chromosome arms and telomeres. Dispersal of a particular satellite is accompanied by changes in sequence structure (for example, base substitutions, deletions, etc.) and a corresponding decrease in its detectability at either the molecular or cytological level. On the basis of this model, observed satellites in newt species may be classified as 'old', 'young', or of 'intermediate' phylogenetic age. The functions and effects of satellite DNA and heterochromatin at the cellular and organismal levels are also discussed. It is suggested that satellite DNA may have an impact on cell proliferation through the effect of late-replicating satellite-rich heterochromatin on the duration of S-phase of the cell cycle. It is argued that even small alterations in cell cycle time due to changes in heterochromatin amount may have magnified effects on organismal growth that may be of adaptive significance.

Clinical application of organ specific isoamylases.

Since hyperamylasemia with or without abdominal pain is a frequently encountered problem, serum isoamylase analysis in 52 patients was done to see if the organ source of the amylase would be helpful in a clinical setting. Four patterns of hyperamylasemia were found: 1) AMY1 (salivary) hyperamylasemia; 2) AMY2 (pancreatic) hyperamylasemia; 3) Both AMY1 and AMY2 amylase elevated; and 4) macroamylasemia. A variety of conditions other than pancreatitis were associated with hyperamylasemia, and some patients who were thought on clinical grounds to have pancreatitis had raised levels of AMY1 (salivary) amylase. This study suggests that hyperamylasemia alone is a poor indicator of pancreatic disease, and that isoamylase analysis will improve the accuracy with which amylase determinations are used.

Aged amylase: a valuable test for detecting and tracking pancreatic pseudocysts.

Amylase-rich fluid that incubates ("ages") within a pancreatic pseudocyst undergoes a change that can be detected by isoenzyme analysis of amylase from the serum. This aging is a result of deamination of the asparagine and glutamine residues on the amylase molecule. Eighteen of 20 patients with surgically proved pseudocysts had greater than 15% aged (deaminated) amylase in their serum. Levels of aged amylase returned to normal following treatment of their pseudocysts. Twenty of 23 patients with acute pancreatitis had levels of aged amylase below 15% (P less than .05). A criterion of 15% aged amylase resulted in 87% specificity, and 91% sensitivity for the diagnosis of pseudocysts. Because this test is noninvasive and easy to perform, it should become the ideal screen for patients at risk of development of pseudocysts, Endoscopic retrograde pancreatography, ultrasonography, and abdominal computed tomographic scanning should be reserved for confirmation of the diagnosis when the result of isoenzyme analysis is positive.