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A better understanding of how bermudagrass (Cynodon spp.) regrowth is influenced by production inputs will aid in advancing precision management. The objectives were to evaluate the effect of irrigation and nitrogen (N) on normalized difference vegetative index (NDVI) during regrowth and evaluate the relationship between NDVI during regrowth to forage yield. Normalized difference vegetative index (NDVI) data were collected in an experiment evaluating two harvest schedules (four or eight week), four rates of irrigation (0, 4.2, 8.4, or 12.5 mm of water each irrigation event), and three rates of N (season total of 168, 336, and 504 kg N ha -1 ). Both irrigation and N influenced NDVI of bermudagrass during the regrowth periods of both the four- and eight-week harvest schedules, but there were no irrigation by N interactions. As was expected, regrowth (as measured by NDVI) in response to irrigation was dependent on the timing and duration of rain-free periods within the regrowth periods. Generally, NDVI increased with increasing N rate at most sampling dates. Regression slopes of yield versus NDVI measured near the end of the regrowth period were higher for the eight-week harvest schedule than for the four-week schedule. Within each harvest schedule, however; slopes were similar. The high correlation coefficients between NDVI late in the regrowth period and yield suggest NDVI is a useful tool for managing bermudagrass harvests.

Ninety plant macrofossil taxa from the Ziegler Reservoir fossil site near Snowmass Village, Colorado, record environmental changes at high elevation (2705 m asl) in the Rocky Mountains during the Last Interglacial Period. Present-day vegetation is aspen forest (Populus tremuloides) intermixed with species of higher (Picea,Abies) and lower (Artemisia,Quercus) elevations. Stratigraphic units 4–13 contain montane forest taxa found near the site today and several species that today generally live at lower elevations within (Abies concolor,Lycopus americanus) and outside Colorado (Najas flexilis). These data suggest near-modern climatic conditions, with slightly warmer summer and winter temperatures. This montane forest period was succeeded by a shorter treeless interval (Unit 14) representing colder and/or drier conditions. In units 15–16, conifer trees reoccur but deciduous and herb taxa are lacking, suggesting a return to warmer conditions, although cooler than during the earlier forest period. Comparison of these inferred paleoclimatic changes with the site's geochronologic framework indicates that the lower interval of sustained warmth correlates with late MIS 6–early 5b (~138–94 ka), the cold interval with MIS 5b (~94–87 ka), and the uppermost cool assemblages with MIS 5a (~87–77 ka).

Death by spinach: They're not words you'd expect to read in an obituary. Yet in 2006, five Americans died and more than 200 others fell ill from eating the salad greens, which were contaminated with a virulent strain of E. coli bacteria.

AIMS--Chromosome 11q23 seems to be a site of frequent mutation in cancer. It also contains loci such as ataxia telangiectasia with possible importance in the pathogenesis of breast tumours. The short arm of chromosome 11 has been studied extensively in breast cancer, but the long arm, in particular the distal part, has been studied less frequently. Cytogenetic analysis has shown possible involvement of chromosome 11q in breast tumours. Chromosome transfer experiments have also implicated chromosome 11q in breast cancer. A high frequency of mutations might therefore be expected to occur on chromosome 11q in breast cancers. METHODS--Using restriction fragment analysis, the primary tumours of 41 patients with breast cancer were screened for mutations at five loci on chromosome 11q (D11Z1, INT2, (FGF3), DRD2, NCAM, and D11S29). RESULTS--Allelic loss occurred at a high frequency (59%) at D11S29. At NCAM, novel alleles were frequently seen on autoradiographs. Relatively low frequencies of mutation were detected at the other loci. Allelic loss at D11S29 was associated with the presence of lymph node metastases, but this may be a chance association. CONCLUSIONS--The frequency of allelic loss at the DS11S29 locus is high. The significance of novel alleles at NCAM and their relation to allelic loss at D11S29 are unclear. The results presented here do not permit fine mapping of a region of allelic loss, but suggest that the region of greatest loss lies distal to DRD2. The results provide further evidence for the importance of gene(s) near 11q23 in the pathogenesis of breast cancer, and of tumours in general.

The mutual climatic range (MCR) technique is perhaps the most widely used method for estimating past climatic parameters from fossil assemblages, largely because it can be conducted on a simple list of the taxa present in an assemblage. When applied to plant macrofossil data, this unweighted approach (MCR un ) will frequently identify a large range for a given climatic parameter where the species in an assemblage can theoretically live together. To narrow this range, we devised a new weighted approach (MCR wt ) that employs information from the modern relations between climatic parameters and plant distributions to lessen the influence of the “tails” of the distributions of the climatic data associated with the taxa in an assemblage. To assess the performance of the MCR approaches, we applied them to a set of modern climatic data and plant distributions on a 25-km grid for North America, and compared observed and estimated climatic values for each grid point. In general, MCR wt was superior to MCR un in providing smaller anomalies, less bias, and better correlations between observed and estimated values. However, by the same measures, the results of Modern Analog Technique (MAT) approaches were superior to MCR wt . Although this might be reason to favor MAT approaches, they are based on assumptions that may not be valid for paleoclimatic reconstructions, including that: 1) the absence of a taxon from a fossil sample is meaningful, 2) plant associations were largely unaffected by past changes in either levels of atmospheric carbon dioxide or in the seasonal distributions of solar radiation, and 3) plant associations of the past are adequately represented on the modern landscape. To illustrate the application of these MCR and MAT approaches to paleoclimatic reconstructions, we applied them to a Pleistocene paleobotanical assemblage from the western United States. From our examinations of the estimates of modern and past climates from vegetation assemblages, we conclude that the MCR un technique provides reliable and unbiased estimates of the ranges of possible climatic conditions that can reasonably be associated with these assemblages. The application of MCR wt and MAT approaches can further constrain these estimates and may provide a systematic way to assess uncertainty. The data sets required for MCR analyses in North America are provided in a parallel publication.

C-type particles are found in human, baboon, marmoset, rhesus, patas, and cynomolgus monkeys. A cebus monkey and two chimpanzee placentas have failed thus far to show the presence of these particles. Of the nonprimate tissues examined one mouse and one guinea pig placenta were positive whereas a rabbit placenta was negative. Variations in number of particles present in tissues as well as number of animals of a given species containing particles are noted. The isolation of foamyviruses from placenta cultures makes critical evaluation and interpretation of findings imperative. Several considerations suggest that the presence of foamyviruses is coincidental.

Well versed in natural history, particularly geology and ornithology, Hugh Edwin Strickland (1811–53) became fascinated by the dodo and mankind's influence on its extinction. Seeking to investigate this flightless bird and other extinct species from islands in the Indian Ocean, he invited the comparative anatomist Alexander Gordon Melville (1819–1901) to help him separate myth from reality. Divided into two sections, this 1848 monograph begins with Strickland's evaluation of the evidence, including historical reports as well as paintings and sketches, many of which are reproduced. Melville then analyses the osteology of the dodo and Rodrigues solitaire, describing his findings from dissections of the few available specimens and making comparisons with similar species. A seminal work, it correctly concluded that the dodo was more closely related to pigeons than vultures, and the book also inspired others to take up the search for new fossil evidence.

The JB6 mouse epidermal cell system has been used extensively as an in vitro transformation model for the study of tumor promotion. The standard JB6 cell assay for promotion of transformation is carried out in soft agar or other anchorage independent conditions. The present study was directed to the development of an in vivo model to distinguish the promotion resistant (P-) and promotion sensitive (P+) progression phenotypes. Results indicate that the grafting assay distinguishes P- and P+ cells in vivo with P+ but not P- cells forming tumors within 7-9 weeks. Expression of dominant negative mutant jun TAM67 blocks both anchorage independent transformation response and graft bed tumor formation by P+ cells, suggesting that the requirement for AP-1 activation in transformation now applies in vivo. Expression of mutated p53 produced a gain of P+ phenotype in P- cells in vitro, but not in vivo. Histochemical and Northern blot analysis for expression of various keratinocyte markers revealed no evidence for expression, suggesting a loss of keratinocyte markers following establishment in culture. In summary, the skin-grafting assay described in this study appears to be a valid in vivo assay for distinguishing the preneoplastic progression phenotypes represented by JB6 P- and P+ cells.

Cell line SCR722 was derived from adult SENCAR mouse epidermal cells initiated in culture by treatment with the carcinogen N-methyl-N′-nitro-N-nitrosoguanidine and selection for foci proliferating in medium with calcium levels that induce terminal differentiation in normal cells. Expansion of one of these foci and two additional cell clonings produced cell line SCR722, which was near-tetraploid and formed normal skin when grafted to athymic nude mouse hosts. However, unlike normal keratinocytes, SCR722 cells fail to suppress papilloma formation when grafted along with papilloma cell line SP-1. For optimum growth in culture, SCR722 cells required fibroblast-conditioned medium and 0.5 mM Ca2+. SCR722 cells had a wild-type c-Ha-ras gene but had lost their requirement for conditioned medium in culture and produced dysplastic papillomas in grafts when transduced with the v-Ha-ras gene. SCR722 cells stably expressing the v-fos gene produced normal epidermis in grafts, but when these cells were transduced with the v-Ha-ras gene, they produced carcinomas. Clones with greater expression of the transfected v-fos gene had a more invasive phenotype in vivo. These results indicate that carcinogen treatment of epithelial cells can result in an altered but nontumorigenic phenotype that may be at risk for becoming a more advanced neoplastic state with additional genetic alterations. © 1995 Wiley-Liss. Inc.

SENCAR mice, developed by selective breeding for high susceptibility to skin carcinogenesis by initiation with 7,12-dimethylbenz[a]anthracene and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA), form squamous papillomas in approximately 20% of animals treated repeatedly with TPA, without chemical initiation. DNA from eight skin tumors produced by a TPA-only protocol and four cell lines derived from these tumors was amplified by polymerase chain reaction and analyzed by discriminative oligonucleotide hybridization using oligomers specific for various c-rasHa gene codon 61 sequences. Five tumors and three cell lines had CAA (wild-type) to CGA mutations. In addition, one tumor had a CAA to CTA mutation, for a total of six of eight tumors having an activating mutation at this codon. Two tumors and one cell line had no codon 61 mutations detectable by this method. Since tumors derived from promotion-only protocols presumably originated from constitutively initiated cells, we examined tumor-free skins of untreated newborn and eight-month-old retired breeders and of 78-88-week-old SENCAR mice of both sexes, which were treated with TPA for 10 weeks starting at age 16-28 weeks and were untreated thereafter. Only the wild-type c-rasHa gene codon 61 sequence was seen, suggesting that the constitutively initiated cell population, if present, is below the limit of detection by this method.

Carcinogenesis in mouse skin can be divided into three distinct stages: initiation, promotion, and progression (malignant conversion). Initiation, induced by a single exposure to a genotoxic carcinogen, can result from a mutation in a single critical gene (e.g., rasHa), apparently in only a few epidermal cells. The change is irreversible. Promotion, resulting in the development of numerous benign tumors (papillomas), is accomplished by the repeated application of a nonmutagenic tumor promoter. The effects of single applications of tumor promoters are reversible since papillomas do not develop after insufficient exposure of initiated skin to promoters or when the interval between individual promoter applications is increased sufficiently. The reversibility of promotion suggests an epigenetic mechanism. Promoter treatment provides an environment that allows the selective clonal expansion of foci of initiated cells. The conversion of squamous papillomas to carcinomas (termed progression or malignant conversion) occurs spontaneously at a low frequency. The rate of progression to malignancy can be significantly increased by treatment of papilloma-bearing mice with certain genotoxic agents. These progressor agents or converting agents are likely to act via a second genetic change in papillomas already bearing the initiating mutation. Progression in the skin is characterized by genetic changes that result in several distinct changes in the levels or activity of structural proteins, growth factors, and proteases. The mechanisms involved in progression are being studied in epidermal cell culture. In order to determine the in vivo phenotype of cultured cells, a grafting system was developed in which the cells were transferred from culture to a prepared skin bed in athymic mice. Introduction of an activated v-fos oncogene into initiated cells bearing an activated rasHa gene produced cells with a carcinoma phenotype, i.e., carcinomas formed when the cells were grafted as part of reconstituted skin. Grafted keratinocytes containing the rasHa gene alone produced papillomas; with v-fos alone, normal skin formed when grafted. The rasHa/fos carcinomas showed changes in differentiation markers characteristic of chemically induced carcinomas. A cell culture assay utilizing cells initiated by the introduction of an activated rasHa oncogene was developed to study progression. After exposure of initiated cells to progressor agents under conditions in which the proliferation of the rasHa-initiated cells was suppressed, proliferating foci developed, with a good correlation of activity in the assay with activity in the progression stage in vivo. The cell culture assay provides a quantitative model to study chemically induced neoplastic progression and may be useful to identify potential progressor agents.

The induction of cancer on mouse skin by initiation-promotion protocols occurs through stages in which a benign squamous papilloma is an obligate precursor of squamous cell carcinoma. Activation of the Ha-ras gene is sufficient to produce the papilloma phenotype, while additional genetic changes are required for malignant conversion. The introduction of Ha-ras into normal keratinocytes suppresses the expression of differentiation markers, keratin K1 and K10, and loricrin (a cornified envelope precursor) and, to a lesser extent, filaggrin, at the level of transcription. However, cells initiated by Ha-ras express a nonepidermal keratin, K8. The transcription of K8 in these cells is sensitive to the level of medium Ca2+, being abundant in 0.5 mM Ca2+ and not detected in 0.05 mM Ca2+. Epidermal differentiation is regulated by signalling, which involves changes in phosphatidylinositol turnover and intracellular Ca2+. Cells initiated by Ha-ras do not differ from normal keratinocytes in their intracellular Ca2+ response patterns, at least in response to changes in extracellular Ca2+ and serum factors. However, c-Ha-ra keratinocytes have a high basal level of phosphatidylinositol (PI) turnover, which is additive with several other inducers of this pathway, including Ca2+ and aluminum fluoride. Additional studies suggest that high turnover of the PI pathway is incompatible with differentiation-specific gene expression in keratinocytes. We suggest this negative relationship is mediated through elevated diacylglycerol production and chronic down-modulation of protein kinase C. Protein kinase C is known to be essential for expression of differentiation-related genes in keratinocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Heartworm-infected dogs were treated therapeutically with a new heartworm adulticide (melarsomine dihydrochloride, RM 340) and then put on a Strategic Program with treatment every 4 months for clinical prophylaxis to take advantage of the drug's potent activity against 4-month-old immature as well as adult Dirofilaria immitis. Ten random-source dogs with naturally acquired heartworm infections (microfilariae- and antigen-positive) were given melarsomine (2.2 mg kg-1 twice 3 h apart) by i.m. injection in the lumbar muscles to clear their existing infections. They were then placed outdoors (August 1988) in a high-risk area in Georgia (USA) for heartworm transmission and given melarsomine at the same posology every 4 months (Strategic Program) for 12 months as a clinical prophylactic measure. Five nontreated heartworm-naive beagles placed at this site during the same period served as 'controls' to monitor heartworm transmission. After exposure for 12 months, the ten treated and five 'control' dogs were taken indoors and held for 5 months. Microfilaremia and antigenemia levels were monitored in both groups by testing at 4-5 month intervals throughout the study and the intensity of infection was determined at necropsy. Microfilaremia levels in treated dogs dropped dramatically following the initial therapeutic treatment and remained either negative or low. Only two of the five 'control' dogs became microfilaremic, and this occurred near the end of the study. Nine of the ten treated dogs were antigen-negative 4 months after the initial therapeutic treatment, and all of them were antigen-negative at all bleedings thereafter. Four of the five 'control' dogs were antigen-positive at necropsy, and only one of these was positive 4 months earlier. Based on these antigen data, the initial treatment cleared 90% of the dogs of worms, and no worms were detected in any of the treated dogs thereafter. However, it is possible that undetectable immature heartworms were present. Although all of the treated dogs were antigen-negative at necropsy, three of them had a total of eight heartworms, seven of which were clearly immature, as determined by worm length measurements, and the remaining worm was a young adult female that was probably too young to be detected. All of the five 'control' dogs had heartworms (average 7.4; range 1-16), and about half of these worms were clearly immatures. Therapeutic treatment followed by strategic treatment with melarsomine every 4 months during reexposure was at least 89.2% effective overall.(ABSTRACT TRUNCATED AT 400 WORDS)

The microbial alkaloid staurosporine induces responses associated with protein kinase C activation, resulting in terminal differentiation in cultures of both normal and neoplastic mouse epidermal cells. As a cancer chemotherapy model, we treated grafts of mouse epidermal tumor cell lines 308 and SP-1 repeatedly with staurosporine. A dose-dependent inhibition of tumor formation, maximal at 0.025 nmol per treatment, was observed. Higher and lower doses were less effective, suggesting a specific target for staurosporine action. A single, low-dose treatment 2 weeks after grafting also markedly reduced tumor formation. Although in vitro evidence suggests that staurosporine-induced terminal squamous differentiation results from activation of protein kinase C, we found no inhibition of tumor growth in similar studies with the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate. These results indicate that staurosporine is an effective antitumor agent for eradicating squamous cell tumors in vivo.

Introduction of the v-Ha-ras gene into primary epidermal keratinocytes, followed by grafting of these cells to animals, leads to the formation of benign epidermal tumors that resemble papillomas induced chemically by a two-stage carcinogenesis protocol. In this study, we investigated v-Ha-ras-induced papillomas for aberrant expression of type I keratin K13, previously described in 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol -13-acetate (DMBA/TPA)-induced mouse epidermal tumors. Papillomas produced from three independent infection series were removed 3 wk after grafting concomitant with control grafts originating from mock-, neo-, and v-fos-infected primary keratinocytes. Combined analysis of the grafts by western blotting of extracted keratins and immunofluorescence studies of frozen sections with a K13-monospecific antibody revealed K13 expression in all v-Ha-ras-induced papillomas and absence of this keratin in all control grafts. K13-positive cells in papillomas were restricted to the suprabasal cell layers of the lesions and, at this stage of papilloma development, occurred as foci of varying extensions. Analysis of genomic DNA from v-Ha-ras-induced papillomas for the methylation state of a CpG dinucleotide in the distant promoter region of the K13 gene revealed the occurrence of unmethylated DNA copies that were generated at the expense of methylated DNA copies ubiquitously present in normal epidermis. The ratio of unmethylated to methylated DNA copies correlated with the extent of suprabasal K13 protein expression. Thus, all features of aberrant K13 expression previously described in DMBA/TPA-induced papillomas were shared by v-Ha-ras-induced papillomas.

Multiple benign squamous papillomas commonly precede the development of an occasional squamous cell carcinoma in mouse skin carcinogenesis. The incidence of carcinomas can be enhanced by treating papilloma-bearing mice with mutagens such as urethane, nitroquinoline-N-oxide, or cisplatinum. This observation suggests that a genetic change is required for malignant conversion. The malignant phenotype is characterized by a marked reduction in the transcription of specific epidermal differentiation markers, a pattern which is useful for the early diagnosis of malignant conversion. Cells expressing a benign phenotype can be obtained by introducing the v-rasHa oncogene into cultured epidermal cells by a replication-defective retrovirus. Alternatively, benign tumor cells can be cultured from papillomas induced by chemical carcinogens in vivo or from carcinogen-treated mouse epidermis. In all cases, the benign phenotype in vitro is characterized by an altered biological response to changes in extracellular calcium, an important determinant of the differentiation state of cultured normal keratinocytes. Transfection of cloned plasmid DNA into benign tumor cells has revealed that transforming constructs of the fos oncogene induce malignant conversion, whereas myc and adenovirus E1A oncogenes do not. The fos carcinomas do not express differentiation-specific epidermal markers and secrete proteases such as transin and urokinase, a set of characteristics previously noted for chemically induced skin carcinomas. Cultured normal epidermal cells, exposed to the v-ras and the v-fos oncogenes simultaneously, are malignantly transformed. Alone, the fos oncogene does not detectably alter the phenotype of normal keratinocytes. These studies indicate that a limited number of genes is involved in epidermal carcinogenesis.

Grafting experiments show that the enhanced sensitivity of the SENCAR mouse to skin carcinogenesis by initiation and promotion is a property of the skin itself, suggesting the usefulness of in vitro studies to elucidate the mechanism. Such studies have indicated that cultured epidermal cells of SENCAR mice and the resistant BALB/c strain are remarkably similar in a variety of respects. DNA repair and carcinogen binding are quantitatively similar in cultured cells of SENCAR and more resistant mouse strains. Epidermal Langerhans cell (LC) number and LC-mediated functions were indistinguishable in SENCAR and BALB/c mice. Primary epidermal cells cultured in the presence of various concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid, epidermal growth factor (EGF), hydrocortisone, or fluocinolone acetonide failed to reveal differences in growth between BALB/c and SENCAR cells. Cells from these animals bound comparable amounts of EGF with similar kinetics, and the modulation of this binding by TPA and retinoic acid was indistinguishable between strains. Spontaneous expression of infectious, endogenous xenotropic type C RNA virus at very low levels could be demonstrated in primary BALB/c epidermal cells and both BALB/c and SENCAR epidermal lines resistant to Ca2+-induced terminal differentiation. The number of foci of initiated cells after exposure to carcinogens in vivo or in vitro did not differ significantly between SENCAR and BALB/c, suggesting that SENCAR sensitivity is primarily to promotion. However, there are qualitative differences between SENCAR and BALB/c foci. The appearance of foci of cells resistant to terminal differentiation in untreated SENCAR cultures supports the evidence from in vivo studies for the existence of a constitutively initiated cell population in SENCAR mouse skin.

SummaryKinetic studies using hybridization with complementary DNAs synthesized from high molecular weight type C viral RNAs showed that virus-specific sequences were among the earliest nonribosomal RNAs to accumulate in the mouse uterus after estrogen treatment. Following hormone administration to ovariectomized animals, significant increases above the low level of viral-specific RNA continuously present in the uterus were observed as early as 4 hr, with maximum levels at 8–16 hr. Subsequently, there was a decrease in the relative concentration of viral RNA. However, levels were still higher than basal at 24 hr, the last time point examined. The increase in viral RNA occurred before a detectable increase in protein synthesis, suggesting that this viral-specific RNA was probably mRNA.We thank Thomas Paisley and Carl Reed for excellent technical assistance with this study and Jean McCammon and Audrey Brammer for preparing the manuscript.

This report confirms the presence of an endogenous Type C virus in the adult NIH Swiss mouse uterus by direct electron microscopy and describes quantitative changes in its expression during pregnancy. Virgin NIH Swiss mice were killed at specific times during the estrous cycle or after mating. Uterine tissues were prepared for p30 analysis immunofluorescence or electron microscopy. Details of methods used are given. Viral p30 levels in the mouse uteri during the estrous cycle did not differ significantly although there was some higher expression during the follicular phase of ovarian activity than during the luteal phase. Following coitus total uterine p30 virus levels increased (p less than .05) about 2-fold; at the terminal week of pregnancy it increased to 10-fold (p less than .05). Following parturition there was a rapid loss. The intensity of fluorescence staining was greatest in the epithelia of pregnant uteri. Electron microscopy in 8 of 18 specimens showed the presence of budding and mature Type C virus particles in the endometrium. Budding was most prevalent near the basal lamina of the epithelial cells. Pregnancy therefore modified expression of Type C RNA virus in the uteri. Variation in the level of estrogens is thought to be the cause. The natural occurrence of such particles reported in a wide variety of mammals suggests that the Type C particle may have a normal physiological function. Observations suggest that p30 elevations observed during the early and late stages of pregnancy are independent of complete viral packaging. Tissue cultures and other methods are being used to determine whether such viruses in very low titers are always present. Their role in biology may then be determined.

SummaryEffects of estrogen administration were compared in young (2-5 months) and aged (2.0-2.5 years), ovariectomized NIH Swiss mice. Two murine leukemia virus proteins, p30 and RNA-directed DNA polymerase, were markedly elevated by estrogen in the uteri of young, ovariectomized mice, but behaved differently in aged animals. The polymerase was low or absent in the uteri of aged mice and showed no increase in response to estrogen. In contrast, p30 responded to estrogen in the aged animals much the same as in the younger mice. This implies that the production of these viral proteins is under separate control mechanisms.The authors gratefully acknowledge the excellent technical assistance of Glenn He-gamyer, Carl Reed, Cindy Lin, and Leon Culler. We thank Ruby Howard for histo-logical preparations, and Dr. Richard Olsen for histological evaluation of our tissue specimens.

SummaryTreatment of ovariectomized NIH Swiss mice with estrogens elevated the level of the murine leukemia virus group specific protein and the activity of an RNA-di-rected DNA polymerase in the uterus. The extent that these markers were raised was dependent on the relative biological potency of the estrogen and on the time interval following treatment. Increases in the levels of both viral marker proteins were evident within 24 hr of treatment and were highest at 48 hr. Subsequently, viral protein levels declined to pretreatment levels.The authors acknowledge the technical assistance of Carl Reed, Carol Lipson, Leon Culler, Sara Myers, and Philip Errico. Antisera used in this study were prepared by Dr. R. Wilsnack, Huntington Research Center, Baltimore, MD.

Antiserum to the DNA polymerase of spleen necrosis virus, a reticuloendotheliosis (RE) group virus, inhibited the polymerase activity of reverse transcriptases from two RE group virus isolates and from mammalian type C retroviruses of murine, feline, and primate origin, but did not inhibit reverse transcriptases of avian myeloblastosis virus (AMV) or the type B or type D mammalian retroviruses tested. Conversely, antiserum to DNA polymerase of a mammalian type C retrovirus, Rauscher murine leukemia virus, inhibited the polymerases of mammalian type C viruses and a RE group virus but was ineffective against AMV polymerase.

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Studies of the mutagenic action required for specific chemicals to produce benign or malignant tumours suggest that in mouse skin at least two genetic events occur before carcinoma formation. The isolation of an activated form of the c-rasH gene from skin papillomas has provided evidence that this gene may be a target for the first mutation, which could constitute the initiating mutation in skin carcinogenesis. In vitro studies indicate that the v-rasH gene of Harvey murine sarcoma virus (Ha-MSV), a replication-defective transforming retrovirus, could impart a conditional initiated phenotype on cultured keratinocytes by blocking their ability to differentiate terminally and arresting them in a late basal cell stage of maturation. We now show that when the Ha-MSV v-rasH gene is introduced into cultured keratinocytes by a defective retroviral vector, skin grafts constructed with cells carrying the mutated ras oncogene produce papillomas on athymic nude mouse recipients. Furthermore, the expression of the exogenous oncogene seems to be regulated at the transcriptional level in the differentiated portions of the benign tumour.

(1844). XXXIX.—On the evidence of the former existence of Struthious birds distinct from the Dodo in the islands near Mauritius. Annals and Magazine of Natural History: Vol. 14, No. 92, pp. 324-326.

Mouse skin papillomas and squamous cell carcinomas induced by initiation with 7,12-dimethylbenz[a]anthracene and promotion with phorbol esters, such as 12-O-tetradecanoyl phorbol-13-acetate, frequently contain an activated Harvey ras gene. Six murine epidermal cells lines established from pooled skin papillomas previously tested negative in the NIH-3T3 assay, but have an altered differentiation program by a variety of criteria. The Harvey ras gene and its p21 protein product from these cell lines have been analyzed for alterations responsible for their altered growth and differentiation properties that were undetectable by 3T3 transfection assays. In comparison with primary papillomas and carcinomas, shown to have a point mutation in codon 61 of the Harvey ras gene, resulting in a p21 product with the diagnostic alteration in SDS-PAGE, the papilloma cell lines exhibited neither the codon 61 mutation, nor p21 product with altered migration in SDS-PAGE. These findings suggest that these papilloma cell lines contain a genetic lesion(s), other than Harvey ras activation, that may be responsible for their altered epithelial differentiation patterns and thus may serve as a useful model for identifying lesions involved in malignant conversion.

The expression of oncornaviral-specific RNA was studied in the NIH Swiss mouse uterus during gestation using hybridization with complementary DNAs synthesized from type C viral high molecular weight RNAs. Following conception there was a marked decrease in the relative concentration of viral RNA below the levels in nonpregnant uteri. Thereafter, the level of viral RNA remained low (less than 30% of prepregnancy levels) until Day 14. Following Day 14 there was a progressive increase in levels of viral RNA and peak levels (greater than or equal to pregestation) were reached just before parturition (Day 18). One day after parturition viral RNA levels were still elevated. A similar profile was observed for p30, a viral-coded protein. We suggest that oncornaviral gene expression is under transcriptional control, most likely by ovarian steroid hormones, and that oncornaviruses can be useful tools to study gene expression in the mammalian uterus. BIOLOGY OF REPRODUCTION 26, 806-812 (1982)

A virus (M-7) isolated from baboon placental tissue demonstrates many similarities to endogenous feline virus RD-114. Immunodiffusion analysis shows a group-specific antigen (gs-1) line of identity between M-7 and RD-114. Anti-RD-114 DNA polymerase IgG inhibits M-7 polymerase by 57% compared to 97% for RD-114. M-7 virus has helper activity as demonstrated by rescue of murine sarcoma virus (MSV) from sarcoma-positive leukemia-negative human amnion cells. The host range of the rescued M-7 pseudotype of MSV, MSV (M-7), is similar to that of RD-114 virus. MSV (M-7) is also able to transform baboon cells and causes no detectable transformation of feline cells without addition of helper feline leukemia virus. Interference properties of M-7 and RD-114 virus are identical. Virus-specific neutralizing antisera, although partially cross-reacting, can distinguish MSV (M-7) from MSV (RD-114). These similarities and differences between RD-114 and M-7 viruses are best explained as type-specific differences between two viruses within the same strain.

Xenotropic type C retraviruses were isolated from cell-free uterine extracts of normal adult NIH Swiss mice by direct inoculation of mink, cat, and human target cells. Successful isolation occurred in approximately one-half the susceptible target cell cultures inoculated with a given extract. These results demonstrate the presence of fully infectious xenotropic virus particles in the uterus of the normal NIH Swiss mouse.

The activation of the c-Ha-ras gene and its contribution to the tumorigenic phenotype were examined in cultured mouse keratinocytes and squamous tumors using transfection into NIH 3T3 cells and nucleic acid hybridization. When normal keratinocytes were cultured in medium with 0.05 mM Ca2+ (low Ca2+ medium), many cells died within 2-3 wk, while others formed rapidly growing foci that could be subcultured. These rapidly growing cells produced benign tumors when grafted to nude mice and possessed a heterozygous mutation in the c-Ha-ras gene with an A----T transversion in codon 61. Fibroblast-conditioned low Ca2+ medium prevented cell death, focus formation, c-Ha-ras gene mutation, and tumorigenicity. Thus, suboptimal culture conditions favored a spontaneous mutation in codon 61 of the c-Ha-ras gene of keratinocytes. When a v-Ha-ras gene was introduced into normal keratinocytes by a replication-defective retrovirus, the recipient cells produced papillomas in vivo, and after 2 mo, 60% of the tumors converted to squamos cell carcinomas. None of the 22 converted tumors had an endogenous c-Ha-ras gene mutation at codon 61. However, the A----T transversion mutation developed when these carcinoma cells were cultured in low Ca2+ medium but not in fibroblast-conditioned medium. Cells with both an exogenous v-Ha-ras and an activated c-Ha61-ras gene produced undifferentiated, rapidly lethal carcinomas, while cells with only v-Ha-ras maintained the squamous carcinoma phenotype. Undifferentiated carcinomas also developed when the v-Ha-ras gene was introduced into papilloma cells with a chemically induced endogenous c-Ha61-ras gene mutation. These results suggest that mutation in the c-Ha-ras gene can contribute to initiation, malignant conversion, and malignant progression in skin carcinogenesis, and gene dosage may determine the phenotype expressed.

To investigate the role of transforming growth factor alpha (TGF alpha) in tumor development, we introduced the human TGF alpha (hTGF alpha) cDNA into cultured primary mouse epidermal cells or papilloma cells using a replication-defective retroviral vector and analyzed skin grafts constructed with such cells. Expression of the exogenous gene was confirmed by detection of hTGF alpha mRNA by northern RNA blot analysis, and the secreted hTGF alpha was measured by ELISA of culture supernatants. Tumor cells expressing hTGF alpha produced benign tumors (papillomas), which were 1.5- to 2-fold larger than tumors of parental cells when tested as skin grafts on nude mice. Grafts of normal cells that expressed hTGF alpha produced normal skin. When mixtures of parental tumor cells and normal mouse keratinocytes were grafted to nude mice, papilloma formation was suppressed and tumors that did form were small. Grafts of hTGF alpha-producing papilloma cells combined with either normal epidermal cells or hTGF alpha-producing epidermal cells yielded large tumors. Mixed grafts containing keratinocytes expressing hTGF alpha and parental papilloma cells also produced large tumors. While the tumor size was substantially increased by hTGF alpha expression, the tumors that developed in all groups were histologically benign and reached a stable size 4-5 wk after grafting. These results indicate that expression of hTGF alpha by either tumor cells (autocrine) or adjoining normal cells (paracrine) can stimulate tumor growth, particularly when tumor growth is suppressed by normal tissue. However, expression of this growth factor did not appear to influence tumor progression directly.

We have used tRNAs protected against periodate oxidation by charging with specific amino acids to show that Escherichia coli valyl-tRNA synthetase ( l -valine : tRNA ligase (AMP), EC 6.1.1.9) can charge several Neurospora crassa tRNAs, including tRNAPhe. Maximum heterologous charging required the presence of dimethylsulfoxide and large concentrations of synthetase. In a mixed N. crassa tRNA preparation, E. coli valyl-tRNA synthetase charged primarily tRNAs other than tRNAPhe; but in a preparation of tRNAPhe further purified about 11-fold on benzoylated DEAE-cellulose, tRNAPhe was the predominant tRNA charged. We conclude if a number of tRNAs can be charged in a heterologous system such as this, then such reactions provide a limited insight into the mechanism of specific recognition of tRNAs by aminoacyl-tRNA synthetases.

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(1840). XXIII.—On the true Method of discovering the Natural System in Zoology and Botany. Annals and Magazine of Natural History: Vol. 6, No. 36, pp. 184-194.