Antigen-driven long term-cultured T cells proliferate in vivo, distribute widely, mediate specific tumor therapy, and persist long-term as functional memory T cells.

Mice bearing disseminated syngeneic FBL-3 leukemia were treated with cyclophosphamide plus long term-cultured T cells immune to FBL-3. The cultured T cells for therapy had been induced to grow in vitro for 62 d by intermittent stimulation with irradiated FBL-3. At the time of therapy, such antigen-driven long term-cultured T cells were greatly expanded in number, proliferated in vitro in response to FBL-3, and were specifically cytotoxic. Following adoptive transfer, donor T cells persisting in the host were identified and counted using donor and host mice congenic for the T cell marker Thy-1. The results show that antigen-driven long term-cultured T cells proliferated rapidly in vivo, distributed widely in host lymphoid organs, and were effective in tumor therapy. Moreover, the already rapid in vivo growth rate of donor T cells could be augmented by administration of exogenous IL-2. When cured mice were examined 120 d after therapy, donor L3T4+ T cells and donor Lyt-2+ T cells could be found in large numbers in host ascites, spleen, and mesenteric and axillary lymph nodes. The persisting donor T cells proliferated in vitro, and became specifically cytotoxic in response to FBL-3, demonstrating that antigen-driven long term-cultured T cells can persist long term in vivo and provide immunologic memory.

Human primary myoblast cell cultures from non-diabetic insulin resistant subjects retain defects in insulin action.

Insulin resistance is a predictor of the development of noninsulin-dependent diabetes mellitus (NIDDM) in humans. It is unclear whether insulin resistance is a primary defect leading to NIDDM or the result of hyperinsulinemia and hyperglycemia. To determine if insulin resistance is the result of extrinsic factors such as hyperinsulinemia primary skeletal muscle cell cultures were established from muscle biopsies from Pima Indians with differing in vivo insulin sensitivities. These cell cultures expressed a variety of muscle-specific phenotypes including the proteins alpha-actinin and myosin, muscle-specific creatine kinase activity, and RNA encoding GLUT4, MYF5, MYOD1, and MYOGENIN. Labeled glucose was used to measure the insulin-stimulated conversion of glucose to glycogen in these cultures. The in vivo rates of insulin-stimulated glycogen production (insulin resistance) were correlated with in vitro measures of glycogen production (P = 0.007, r = 0.58). This defect in insulin action is stable in a uniform culture environment and is retained over time. The retention of insulin resistance in myoblast derived cell cultures is consistent with the expression of an underlying biochemical defect in insulin resistant skeletal muscle.

An autosomal genomic scan for loci linked to prediabetic phenotypes in Pima Indians.

Type 2 diabetes mellitus is a common chronic disease that is thought to have a substantial genetic basis. Identification of the genes responsible has been hampered by the complex nature of the syndrome. Abnormalities in insulin secretion and insulin action predict the development of type 2 diabetes and are, themselves, highly heritable traits. Since fewer genes may contribute to these precursors of type 2 diabetes than to the overall syndrome, such genes may be easier to identify. We, therefore, undertook an autosomal genomic scan to identify loci linked to prediabetic traits in Pima Indians, a population with a high prevalence of type 2 diabetes. 363 nondiabetic Pima Indians were genotyped at 516 polymorphic microsatellite markers on all 22 autosomes. Linkage analyses were performed using three methods (single-marker, nonparametric multipoint [MAPMAKER/SIBS], and variance components multipoint). These analyses provided evidence for linkage at several chromosomal regions, including 3q21-24 linked to fasting plasma insulin concentration and in vivo insulin action, 4p15-q12 linked to fasting plasma insulin concentration, 9q21 linked to 2-h insulin concentration during oral glucose tolerance testing, and 22q12-13 linked to fasting plasma glucose concentration. These results suggest loci that may harbor genes contributing to type 2 diabetes in Pima Indians. None of the linkages exceeded a LOD score of 3.6 (a 5% probability of occurring in a genome-wide scan). These findings must, therefore, be considered tentative until extended in this population or replicated in others.

Linkage analysis of acute insulin secretion with GLUT2 and glucokinase in Pima Indians and the identification of a missense mutation in GLUT2.

The acute insulin response (AIR), a measure of pancreatic beta-cell function, aggregates in families and is a predictor for the development of non-insulin-dependent diabetes mellitus (NIDDM) in insulin-resistant Pima Indians. To assess the genetic components of AIR and NIDDM, polymorphic dinucleotide repeat regions in two candidate genes, the liver/islet glucose transporter gene (GLUT2) and the glucokinase gene, were evaluated. Sib-pair linkage analyses were performed to determine if linkage exists between these marker loci and measurements of AIR and NIDDM. No linkage was found between glucokinase and either AIR or NIDDM. Robust sib-pair linkage analyses suggest linkage between GLUT2 and acute insulin response (P = 0.04), but no linkage was observed with NIDDM. The coding region of the GLUT2 gene was screened for mutations using polymerase chain reaction-single-strand conformation polymorphism analysis. A single base change was identified in exon 3 in approximately 5% of the study population, and it constitutes the first reported mutation in the human GLUT2 gene. This base change resulted in an amino acid substitution (Thr110-->Ile110) in the second membrane-spanning region of the GLUT2 protein. No significant association was noted between AIR and the presence or absence of the mutation. Thus, this mutation in GLUT2 is unlikely the cause of a low AIR in Pima Indians.

Evidence for linkage between a region on chromosome 1p and the acute insulin response in Pima Indians.

A low acute insulin response (AIR) is a predictor of non-insulin-dependent diabetes mellitus (NIDDM) in insulin-resistant Pima Indians. We have initiated a search for regions of the genome linked with the AIR using sib-pair linkage analysis as a first step in identifying genes that are determinants of this phenotype. Eighteen short tandem-repeat polymorphisms from chromosome 1 were genotyped in over 900 Pima Indians and tested for linkage with NIDDM and in a subset of Pima Indians for linkage with AIR. The anonymous DNA marker D1S198 on chromosome 1p was linked with AIR (P = 0.000056) in 175 sib pairs from 60 families, all with normal glucose tolerance, but no linkage was observed between D1S198 and NIDDM (P = 0.44, 996 sib pairs). Additional markers genotyped on chromosome 1 did not show linkage with AIR or NIDDM. This study indicates that a locus on chromosome 1p may be a determinant of the phenotypic variation seen in the AIR.

Absence of linkage of obesity and energy metabolism to markers flanking homologues of rodent obesity genes in Pima Indians.

The homologues of single genes that cause obesity in rodents are suggested as candidate genes for modulation of body composition in humans. Among these genes are the four mouse mutations-diabetes (db), obesity (ob), tubby (tub), and yellow agouti (Ay). Variation in the human counterparts to these genes (OB, DB, TUB, and ASP, respectively) may contribute to human obesity, which is thought to have a substantial genetic component. To initially assess the potential contribution of these genes to human obesity, we examined polymorphic DNA markers that, by virtue of syntenic relationships to appropriate regions of the mouse genome, should be closely linked to the human counterparts of these genes. Using combined data from 716 Pima Indians comprising 217 nuclear families, we have tested a number of polymorphic microsatellite markers (three at DB, two at OB, five at TUB, and three at ASP) for sib-pair linkage to BMI, percentage body fat, resting metabolic rate, 24-h energy expenditure, and 24-h respiratory quotient. No significant linkages were found in an analysis of all sibships or in an analysis restricted to discordant sib pairs.

Insulin regulation of multiple ribonucleic acid species in human skeletal muscle in insulin-sensitive and insulin-resistant subjects.

In vivo short term (2 h) insulin-regulated gene expression was examined in skeletal muscle of persons with differing insulin sensitivities. Nine genes were analyzed by a S1 nuclease protection assay with multiple probes (multiple S1 nuclease protection assay) to allow the simultaneous examination of RNA abundances from the multiple genes. In insulin-sensitive individuals, 5 of these 9 genes were insulin responsive. RNA from the proto-oncogenes c-Ha-ras, c-myc, and c-src transiently increased 2- to 4-fold within 30 min of insulin infusion. In addition, the RNA abundance of myf-5, a muscle specific differentiation factor, increased 3-fold with a time course similar to that of c-Ha-ras, c-myc, and c-src. In contrast, type 1 protein phosphatase alpha (PPP1A) RNA levels decreased by 50% within 30 min. In insulin-resistant individuals, the RNA levels of c-Ha-ras and myf-5 did not increase, whereas c-src RNA did increase within 30 min of insulin infusion. RNA encoding c-myc transiently increased in both groups; however, this response was lower in insulin-resistant individuals than in insulin-sensitive individuals in a pattern similar to c-Ha-ras and myf-5. PPP1A RNA levels slightly increased in insulin-resistant individuals. In both insulin-sensitive and insulin-resistant persons, RNA quantities of GLUT4, c-jun, c-fos, and the insulin receptor did not change over the period of insulin infusion. However, overall RNA levels of the insulin receptor and c-jun were lower in insulin-resistant individuals.

Genetic abnormalities in sporadic parathyroid adenomas: loss of heterozygosity for chromosome 3q markers flanking the calcium receptor locus.

Inactivating mutations of the parathyroid cell calcium receptor (CaR) gene cause one form of familial benign/hypocalciuric hypercalcemia, and in homozygous form, cause neonatal severe primary hyperparathyroidism with parathyroid hyperplasia. Thus, we postulated that partial or total loss of CaR function might contribute to calcium insensitivity or even stimulate cell proliferation in sporadic parathyroid adenomas (PAds). To examine this possibility, we sought loss of heterozygosity (LOH) for markers flanking the CaR locus (3cen-3q21) in 35 PAds. We used 16 highly-polymorphic PCR-based markers in paired normal and tumor DNA, extracted from slices of archived surgical specimens. Nineteen to 24 of the DNA pairs were informative with at least one marker. In two informative pairs, we found LOH for markers D3S1303, D3S1267, or D3S1269, which are tightly-linked with and flank the CaR locus. In one tumor, deletion mapping confined the lost area between D3S1271 and D3S1238 (41.7 centimorgans, cM). In the other tumor, LOH spanned most of chromosome 3, ranging at least from D3S1307 to D3S1311 (271.4 cM). LOH was confirmed by repetition of the experiments and quantified by phosphorimaging. Thus, we found LOH encompassing the CaR locus in approximately 10% of sporadic PAds. These data are consistent with the hypothesis that loss of CaR function may occur in PAds, with functional consequences for calcium sensitivity and cell proliferation.

Sex differences, sex ratios and sex roles.

Sexual selection theory predicts that sex roles will be determined by the operational sex ratio (OSR), the sex ratio among individuals searching for mates at any given time. There are two predictions: (i) the sex which is in 'excess' will be the more competitive sex with respect to access to mates; and (ii) the sex of which there is a 'shortage' will be the more choosy with respect to potential partners. We examine the second prediction and find that current OSR theory does not consider an important factor which affects mate choice. This factor is sex differences in variation in mate quality. Hence, we develop a new model of mate choice which shows that the parameter which should be optimized during mate choice is the trade-off between reproductive rate and mate quality. If mate choice is too lax, reproductive rate may be high but partners will be of low quality. If mate choice is too stringent, partners will be of high quality but reproductive rate will be low because such partners will be rare. Stringency of mate choice is, therefore, a facet of OSR theory. Indeed, our model shows that OSR theory can be used to integrate the effect of sex differences in both mating rate and variation in mate quality to predict the direction of mate choice. Our model suggests that: (i) mate choice is only selected when individuals of the opposite sex vary in their quality as mates; (ii) if the extent of variation in mate quality is equal within each sex, the sex with the lower potential mating rate will be the more choosy sex; but (iii) if there is sufficiently greater variation in mate quality among the sex with the lower potential reproductive rate, the sex with the higher potential mating rate will be the more choosy sex.(ABSTRACT TRUNCATED AT 250 WORDS)

Linkage between stature and a region on chromosome 20 and analysis of a candidate gene, bone morphogenetic protein 2.

Sib-pair linkage analysis of the quantitative trait, structure, in over 500 Pima Indians indicates that a genetic determinant of governing stature is located on chromosome 20. Analysis of 10 short tandem repeat polymorphisms localized this linkage to a 3.2cM region that includes D20S98 and D20S66. Using all possible sib-pair combinations, linkage was detected to both stature (P = 0.0001) and to leg length (P = 0.001), but not to sitting height. Single-strand conformational polymorphism analysis of exon 3 of the bone morphogenetic protein 2 (BMP2) gene, a candidate gene in this region, in genomic DNA of 20 of the tallest and 20 of the shortest individuals did not show any consistent differences associated with leg length or height. Sequence analysis of the region encoding the mature protein revealed a single nucleotide substitution, a T to G transversion, not detected by single-strand conformational polymorphism (SSCP) analysis. This transversion results in a conservative amino acid substitution of glycine for valine at codon 80 of BMP2. The frequency of this allele was 0.23 in the sample. No significant differences in height were noted in persons carrying either allele. This indicates that this structural alteration is the mature BMP2 protein does not contribute to the differences in stature observed in the Pima Indians, nor is this structural change in the mature protein likely to be responsible for the linkage observed with stature on chromosome 20.

Spatial organization of a desert rodent community: food addition and species removal.

From 1977 through 1983 we conducted experiments on a desert rodent community where supplemental seeds were added or certain rodent species and ants were removed from 0.25-ha fenced plots in a Chihuahuan Desert site in southeastern Arizona, USA. In this paper we examine the patterns of microhabitat use relative to vegetative cover by 11 rodent species. The results show that: i) removal of the largest seed-eating species, Dipodomys spectabilis, produced the most pervasive and dramatic shifts in microhabitat use by the remaining rodent species; ii) adding seeds or removing ants had little effect on the spatial use of microhabitats by rodents in this community; and iii) non-granivores were just as likely as granivores to shift microhabitat use when other granivores were removed. We believe these results indicate that both food and foraging microsites are limited but the relegation of subdominant species to lesspreferred microhabitats by the large Dipodomys spectabilis is the major factor underlying the spatial organization of this community. Results also demonstrate that strong interactions among species increase the probability that pathways of indirect interactions through intermediary species are important; these complex linkages may include species that overlap little in food preferences.

Seed selection by dark-eyed juncos (Junco hyemalis): optimal foraging with nutrient constraints?

Observations of the foraging behavior of six captive dark-eyed juncos (Junco hyemalis) are used to test the assumptions and predictions of optimal diet choice models (Pyke et al. 1977) that include nutrients (Pulliam 1975). The birds sequentially encountered single seeds of niger thistle (Guizotia abyssinica) and of canary grass (Phalaris canariensis) on an artificial substrate in the laboratory. Niger thistle seeds were preferred by all birds although their profitability in terms of energy intake (J/s) was less than the profitability of canary grass seeds. Of four nutritional components used to calculate profitabilities (mg/s) lipid content was the only characteristic that could explain the junco's seed preference. As predicted by optimal diet theory the probability of consuming niger thistle seeds was independent of seed abundance. However, the consumption of 71-84% rather than 100% of the seeds encountered is not consistent with the prediction of all-or-nothing selection. Canary grass seeds were consumed at a constant rate (no./s) independent of the number of seeds encountered. This consumption pattern invalidates a model that assumes strict maximization. However, it is consistent with the assumption that canary grass seeds contain a nutrient which is required in minimum amounts to meet physiological demands (Pulliam 1975). These experiments emphasize the importance of incorporating nutrients into optimal foraging models and of combining seed preference studies with studies of the metabolic requirements of consumers.

Hydrology and the natural heritage of the Scottish mountains.

The physical natures of the Scottish mountains and their geographical position have created a montane environment, which can be considered as unique in European terms. The mountains of Scotland have been subjected to major environmental changes throughout the past centuries including climate change, deforestation, hydropower developments and more recently the expansion of plantation forestry. Mountain ecosystems have the ability to withstand large climatic variations and extreme events but it is suggested that they may not withstand some of the climatic barriers, which have recently been crossed. The greatest recent land use change in Scotland's mountains has been the expansion of plantation forests. The effects on headwater catchment hydrology are mainly in the reduction in runoff. It is suggested that plantation forestry has a more significant impact on the natural heritage through other influences such as water chemistry and river sediments. Future management of the Scottish mountains needs to consider the great natural heritage value in addition to other interests such as water resources, hydropower generation, commercial forestry and tourism.

Regional and historical variation in the nitrogen content of Racomitrium lanuginosum in Britain in relation to atmospheric nitrogen deposition.

The moss Racomitrium lanuginosum (Hedw.) Brid. is an important component of the drier parts of ombrotrophic mires and montane heaths in north-western Britain. The extent and quality of the montane heaths dominated by R. lanuginosum has declined in recent decades, perhaps in part due to the effects of acidic deposition at high elevations. This paper examines the effect of atmospheric nitrogen deposition, which has increased during this century, on the nitrogen content of R. lanuginosum in Britain. The nitrogen content of the moss reflects the magnitude of the atmospheric supply being least in north-western Scotland and greatest (as much as six-fold greater) near to urban centres in northern England. This regional difference was less marked (only approx. two-fold) during the 19th century (as revealed from the analysis of herbarium specimens) when nitrogen concentrations were appreciably lower. Transplant studies both between regions and between sites within a mountain system demonstrated the importance of atmospheric deposition in determining the tissue nitrogen concentration of the moss. The results are discussed in relation to the potential importance of the enhanced atmospheric nitrogen supply to the normally nitrogen-impoverished montane heaths, and to the growth and persistence of the moss.

Isolation of an adenovirus from an Arab foal with a combined immunode ficiency disease.

Some details of the clinical and postmortem findings of an Arab foal that died as a consequence of adenoviral pneumonia superimposed on a combined immunodeficiency disease are provided. The foal was the 17th in a series of similar deaths that occurred on a farm since 1959. An adenovirus, which by haemagglutination inhibition and serum neutralisation tests was antigenically similar to 2 other equine adenoviruses isolated in Australia, was isolated from a nasal swab taken from the foal when it was 23 days of age.

Inheritance of a lethal immunodeficiency disease of Arabian foals.

A fatal syndrome of certain Arabian foals which begins at about 25 days of age (range 14 to 46 days) and which runs a course of about 23 days (range 13 to 42 days) is described. The syndrome, which affected 17 foals on a single farm is further characterised by pneumonia, in some instances by dermatitis (dermatophilosis) and other infections, together with a progressive decline in health till death at about 49 days of age (range 34 to 77 days), despite intensive therapy. Four of the foals, on histopathological evidence, had adenviral pneumonia, in 2 foals there was histopathological evidence of an immunodeficiency disease and an adenovirus was isolated from 1 foal. It is argued, however, that the 17 foals were affected by a single, specific syndrome (an immunodeficiency disease) and that this disease is inherited as a simple, recessive, autosomal gene.

Regulation of Na(+)-K(+)-2Cl- cotransporter activity in rat skeletal muscle and intestinal epithelial cells.

In mammalian cells, Na(+)-K(+)-2Cl- cotransporter activity participates in regulation of ion and volume homeostasis. This makes NKCC indispensable for normal cell growth and proliferation. We recently reported the existence of two mechanisms that can regulate NKCC activity in mature skeletal muscle. In isosmotic conditions, signaling through ERK MAPK pathway is necessary, while inhibition of the cAMP-dependent protein kinase A (PKA) pathway stimulates NKCC activity during hyperosmotic challenge. Both pathways are involved in regulating cell proliferation in wide variety of cells of epithelial and non-epithelial origin, so we tested which pathway regulated NKCC activity in cultured cells. In cultured rat skeletal muscle (L6) and intestinal epithelial (IEC-6) cells, NKCC activity in the basal state comprised 30 to 50% of total potassium influx, assessed as bumetanide-sensitive 38Rb-uptake. This NKCC activity could not be abolished by inhibitors of ERK MAPK (PD98059 and U0126), PKC (GF109203X), or PI 3-K (wortmannin, LY294002). In L6 myoblasts and in IEC-6 cells, elevation of cAMP levels with isoproterenol or forskolin led to a 60% inhibition on NKCC activity. In contrast, incubation of IEC-6 cells with the PKA-inhibitor H-89 resulted in 50% increase of NKCC activity compared with the basal level. In conclusion, it appears that in cultured cells the cAMP--PKA pathway regulates NKCC activity. This resembles hyperosmotic regulation of NKCC activity.

Identification of Caenorhabditis elegans genes regulating longevity using enhanced RNAi-sensitive strains.

A systematic genome-wide RNA interference screen was performed in the Caenorhabditis elegans lin-15b;eri-1 strain, which has an enhanced response to double-stranded RNA including the nervous system, to identify life-span regulatory factors. In total, 16,757 genes were examined, revealing 115 gene inactivations that extended life span. A more stringent longitudinal analysis revealed 18 gene inactivations that induced the greatest increase in life span (10-90%), all of which extended life span when inactivated either in eri-1 alone or in a second strain with an enhanced response to double-stranded RNA, eri-3. Most reduced the rate of aging, implying that animals aged more slowly. As was the case in previous studies, genes critical for metabolism caused the greatest extension of longevity. Extension of life span occurs through disparate mechanisms as increased resistance to thermal stress, oxidative damage, and decreased age pigment accumulation analysis of the 18 stronger positives failed to demonstrate a correlation between enhanced stress resistance and decreased lysosomal function. Consistently, aps-3 and lys-10, two genes annotated to have lysosomal functions, extended life span when inactivated without enhancing stress resistance. The results of this study reinforce the importance of metabolism, mitochondrial and lysosomal functions, genomic stability, and stress resistance on animal life-span determination.

Assessment of the use and feasibility of video to supplement the genetic counseling process: a cancer genetic counseling perspective.

Cancer genetic counselors use a variety of teaching modalities for patient education. This survey of cancer genetic counselors assessed their use of educational videos and their recommendations for content of future videos. Thirty percent of respondents use videos for patient education. Cited benefits included reinforcement of information for clients and increased counselor efficiency. Of the 70% who do not use videos, predominant barriers included the perceived lack of an appropriate video, lack of space and/or equipment, and concern that videos are impersonal. Most respondents desired a video that is representative of the genetic counseling session, but emphasized the importance of using broad information. Content considered critical included the pros and cons of genetic testing, associated psychosocial implications, and genetic discrimination. The results of this exploratory study provide data relevant for the development of a cancer genetics video for patient education, and suggestions are made based on aspects of information processing and communication theories.