Two sex-linked loci in the leopard frog, Rana pipiens.

Crosses involving one heterozygous parent were performed to test the inheritance of enzymes in the leopard frog, Rana pipiens. After metamorphosis, offspring were sexed and tissue extracts from them were analyzed by gel electrophoresis. Enzyme genotype and sex showed independent assortment for 10 of 12 enzymes heterozygous in the male parent. However, among the offspring of males heterozygous for peptidase C (Pep-C) or superoxide dismutase 1 (SOD-1), male progeny tend to inherit one allele, whereas female progeny tend to inherit the other. Data from several different crosses yield recombination frequencies of 8.6% between sex and SOD-1, 6.9% between SOD-1 and Pep-C and 12.1% between sex and Pep-C. When the female parent is heterozygous for these enzymes no significant difference is seen, in the offspring, between male and female homozygotes and heterozygotes. These results confirm that males are the heterogametic sex in R. pipiens and suggest that sex is determined by a small number of genes on otherwise identical X and Y chromosomes.

Synthesis of certain 4'-substituted nucleosides.

We have developed general methods for the synthesis of 4'-fluoro- and 4'-methoxynucleosides by addition of iodinemonofluoride or iodine and methanol across the double bond of suitably protected 4',5'-unsaturated pyrimidine and purine nucleosides. The structures of these adducts have been determined by a combination of chemical, spectroscopic, and electrophoretic methods. The 4'-methoxy- and the uridine analogs of nucleocidin have been synthesized from the corresponding 4'-fluorouridine and 4'-methoxyadenosine derivatives.

Regeneration of digits and forelimbs in the Kenyan reed frog Hyperolius viridiflavus ferniquei.

Newly metamorphosed individuals of the Kenyan reed frog, Hyperolius viridiflavus ferniquei, are able completely to regenerate amputated digits, including the morphologically complex digital pad. The sequence of morphological events is very similar to that seen in the typical epimorphic regeneration of amphibian extremities. Amputated forearms typically produce hypomorphic spike regenerates. Despite the lack of underlying hand and digital structures, an apical "digital" pad commonly differentiates.

The production of equine monoclonal immunoglobulins by horse-mouse heterohybridomas.

Studies were carried out to determine the optimum conditions for the production of equine monoclonal antibodies (MAbs). Lymphocytes from ponies immunised with influenza A equine 2 virus, isolate A/Equine/Newmarket/79 (H3N8) were fused with mouse myeloma (NSO) cells and with horse-mouse heterohybridomas made aminopterin-sensitive by selective growth in 8-azaguanine. Although all fusions initially resulted in heterohybridoma colonies that secreted equine immunoglobulin, many of these were unable to maintain secretion for longer than a few weeks. Increasing the time between immunisation and the booster injection of Newmarket/79 virus, the inclusion of Freund's incomplete adjuvant and the use of an aminopterin-sensitive primary heterohybridoma as the fusion partner, improved the production of HIg-secreting heterohybridomas. After two clonings eight cell lines were established which maintained anti-Newmarket/79 antibody secretion for over a year. FACS analysis of the cell lines provided a useful means of predicting breakdown of MAb secretion by the cell lines, thus enabling re-cloning to be carried out in time.

The bidirectional relationship between achievement and externalizing behavior problems of students with learning disabilities.

The current study examined the bidirectional relationship between academic achievement and externalizing behavior problems of adolescents with learning disabilities. Forty-three students attending a residential school were assessed for externalizing behavior problems via parent and teacher reports on the Child Behavior Checklist and the Children's Attention and Adjustment Survey. The Wide Range Achievement Test-Revised was used to estimate academic achievement in reading, spelling, and arithmetic. Achievement measures did not predict externalizing behavior problems 1 to 2 years later, although verbal IQ predicted parent reports. Teacher reports of externalizing behavior problems predicted reading and spelling achievement scores 1 year later, and parent reports of externalizing behavior problems predicted changes in reading achievement. There was evidence that attentional problems were the component of externalizing behavior accounting for these relations, consistent with the explanation that inattentive students have difficulty achieving in an intensive learning environment.

The Escherichia coli heat-labile enterotoxin B subunit protects from allergic airway disease development by inducing CD4+ regulatory T cells.

The B subunit of E. coli heat-labile enterotoxin (EtxB) protects against the development of T helper type 1 (Th1)-mediated autoimmune pathologies in mice. Protection was transferable with splenic CD4(+) T cells and was less effective following CD25 depletion; implying a T regulatory cell (Treg)-mediated process. We hypothesized that if this were the case, then EtxB would also control a Th2-mediated disorder. We tested the effect of EtxB treatment on asthma development in ovalbumin (OVA)-sensitized mice. EtxB treatment diminished eosinophilia in bronchoalveolar lavage samples, reduced OVA-specific immunoglobulin E and interleukin 4 production locally and systemically, and reduced airway hyper-reactivity. EtxB induced a dose-dependent increase in Foxp3(+)CD4(+) T cells, and adoptive transfer of splenic CD4(+) T cells partially suppressed lung pathology. Importantly, EtxB treatment increased OVA-specific CD4(+)Foxp3(+) T cells in the lung and systemically. These data demonstrate that EtxB modulates the differentiation of allergen-specific T cells causing inducible Treg induction and preventing disease.

Genetic relationships between feral cattle from Chirikof Island, Alaska and other breeds.

The origin of cattle on Chirikof Island, off the coast of Alaska, is not well documented. We assessed genetic differentiation of cattle isolated on Chirikof Island from several breeds commonly used for commercial production in North America including breeds popularly believed to have contributed to the Chirikof Island population. A set of 34 microsatellite loci was used to genotype Angus, Charolais, Hereford, Highland, Limousin, Red Angus, Salers, Shorthorn, Simmental, Tarentaise and Texas Longhorn cattle sampled from North America and the Chirikof Island population. Resulting F(ST) statistics for these loci ranged from 0.06 to 0.22 and on average, 14% of total genetic variation was between breeds. Whether population structure was modelled as a bifurcating tree or genetic network, Chirikof Island cattle appeared to be unique and strongly differentiated relative to the other breeds that were sampled. Bayesian clustering for multiple-locus assignment to genetic groups indicated low levels of admixture in the Chirikof Island population. Thus, the Chirikof Island population may be a novel genetic resource of some importance for conservation and industry.

A phylogeny for the African treefrog family Hyperoliidae based on mitochondrial rDNA.

Mitochondrial 12S rRNA sequences were used to construct a phylogeny for the African treefrog family, Hyperoliidae. The sequences were aligned using the well-established mt 12S rRNA secondary structure as a map to designate stem and loop positions. Conservation of secondary structure permits a more accurate basis for assessing homologies than does primary sequence alone. The molecular trees showed many similarities to the morphological trees constructed previously. Species within a genus always grouped together. The genera Hyperolius, Heterixalus, Afrixalus, and Kassina were resolved, but not with statistical significance. Leptopelis was the basal group. The analysis shows clearly that Phylyctimantis groups with Kassina as it did in Drewes' morphological tree and that Tachycnemis groups closely with Heterisxalus, a relationship not suggested by the morphological data. Character weighting (including compensatory base changes), mitochondrial trees vs gene trees, and the biogeography of the group are discussed.

Cancer and humiliation: The "catch 22" of disease.

In the author's own words: I am a cancer patient involved in an agonizing struggle against disease. I decided to write about the humiliating aspects of cancer because I have experienced them firsthand. I know them all too well. I have incorporated my feelings about the humiliation of disease ... hoping they may help the reader better understand.

Peptidase isozymes of the leopard frog Rana pipiens: properties and genetics.

The substrate specificity of five peptidases in tissues of the leopard frog, Rana pipiens, corresponds to mammalian peptidases (Pep) A, B, C, D, and S. The inheritance of electrophoretic variants of Pep A, B, C, and D verifies the genetic basis of the variants and the control of the enzymes by separate genetic loci. Electrophoretic patterns of heterozygotes suggest that Pep A, B, and D are dimers, whereas Pep C has a monomer structure. The tissue distribution and developmental patterns suggest that frog peptidases are subject to cellular regulation. Tests of genetic linkage show independent assortment for Pep A and Pep C and for Pep C and Pep D. Pep C is closely linked to superoxide dismutase-1 (6.87% recombination), and Pep B is linked to mannose phosphate isomerase (30.4% recombination).

The occurrence of diploid ova in Rana pipiens.

All 12 Rana pipiens females tested from three populations produced diploid ova. These were identified by the development of from 0.15 to 35 percent per clutch of normal diploid-like embryos among large numbers of haploid embryos following activation of R. pipiens eggs with irradiated R. clamitans sperm. Their diploid nuclear constitution was demonstrated by diploid cell size at Shumway Stage 19, and by the diploid number of chromosomes both as embryos and as mature frogs, and was confirmed by the occurrence of triploid embryos among normally fertilized progeny from the same female parents. Although the precise cytogenetic events leading to the origin of these diploid ova were not directly determined, we were led to conclude that, although diploid ova may result from polynucleate oocytes, the diploidy reported here was an expression of abnormal meiosis that occurred under genetic control. Such anomalies, at the orders of frequency we observed in R. pipiens, have important consequences for all studies using Anuran model systems.

Inheritance of enzymes and blood proteins in the leopard frog, Rana pipiens: three linkage groups established.

Individuals from natural populations of the leopard frog, Rana pipiens, were analyzed for electrophoretic differences in blood proteins and enzymes from an amputated digit. The proteins examined represent products of 72 loci. Presumptive heterozygotes at multiple loci were selected for experimental crosses. Mendelian inheritance of 18 protein variations were demonstrated in the offspring. Tests for linkage or independent assortment were performed for 75 locus pairs. Three linkage groups were established. Linkage group 1 contains two loci, aconitase-1 (Acon 1) and serum albumin (Alb), with a 19% recombination frequency between them. Linkage group 2 contains four loci, glyoxalase (Gly), acid phosphatase-1 (Ap1), acid phosphatase-2 (AP2), and esterase-5 (Est5). The data show the relationships Gly-21.1%-AP1-0%-AP2-6.3%-Est5, and Gly-25.6%-Est5. Linkage group 3 consists of four closely linked esterase loci. The data, Est1-5.1%-Est6, Est6-1.8%-Est10-1.9%-Est4 and Est6-3.0%-Est4, do not establish a complete order but suggest that Est10 is between Est4 and Est6. These results, with data demonstrating apparent independent assortment of 67 other locus pairs, provide a foundation for establishing the frog genetic map.

Genetic and demographic dynamics of small populations of Silene latifolia.

Small local populations of Silene alba, a short-lived herbaceous plant, were sampled in 1994 and again in 1999. Sampling included estimates of population size and genetic diversity, as measured at six polymorphic allozyme loci. When averaged across populations, there was very little change between samples (about three generations) in population size, measures of within-population genetic diversity such as number of alleles or expected heterozygosity, or in the apportionment of genetic diversity within and among populations as measured by F(st). However, individual populations changed considerably, both in terms of numbers of individuals and genetic composition. Some populations doubled in size between samples, while others shrank by more than 75%. Similarly, expected heterozygosity and allele number increased by more than two-fold in individual populations and decreased by more than three-fold in others. When population-specific change in number and change in measures of genetic diversity were considered together, significant positive correlations were found between the demographic and genetic variables. It is speculated that some populations were released from the demographic consequences of inbreeding depression by gene flow.

Enhancement of the immune response to non-replicating herpes simplex virus type-1 preparations by mucosal administration in the presence of cholera toxin.

Different immunization regimes were compared in order to enhance the immune response following mucosal administration of non-replicating HSV-1 preparations to mice. The serum anti-HSV Ig response following intragastric administration of heat or UV inactivated HSV-1 strain SC16 was compared with that elicited by an attenuated derivative of SC16 (TKDM21). The highest response followed immunization with TKDM21 and this was markedly enhanced by repeated intragastric administration, reaching ca 35% of that elicited following a cutaneous infection with live virus. Repeated doses of killed virus produced only a minimal increase in the response even when given intranasally (i.n.). However, cholera toxin and its B-subunit with UV-inactivated virus or a mixture of purified viral glycoproteins enhanced the anti-HSV response after i.n. immunization and produced antibody levels equivalent to those following intragastric delivery of live TKDM21.

Expression of the large plastid gene, ORF2280, in tomato fruits and flowers.

The expression of ORF2280, a large plastid gene of unknown function, was examined in tomato leaves, in a developmental series of tomato fruits, and in tomato flowers. Western blots indicated that much more ORF2280 protein is present in fruits and flowers than in leaves. The most abundant proteins detected, 68 and 59 kDa, are present in about equal amounts in fruits of all stages; they are even more abundant in flowers. A 170-kDa ORF2280 protein is also present in fruit of all stages; it is most abundant in small green fruit. The presence of higher levels of ORF2280 proteins in tomato fruits and flowers indicates that it may have a specialized function in these nonphotosynthetic tissues.

Synteny mapping in the horse using horse-mouse heterohybridomas.

In a study of 35 horse-mouse heterohybridoma cell lines, synteny in the horse was found between LDHB, PEPB and IGF1 and between NP, MPI and IDH2. A synteny between ADA and PEPC was also indicated. The loci for horse immunoglobulin light chain (IgL) genes and for LDHA were independent.

Protection against recurrent ocular herpes simplex virus type 1 disease after therapeutic vaccination of latently infected mice.

The potential of therapeutic vaccination of animals latently infected with herpes simplex virus type 1 (HSV-1) to enhance protective immunity to the virus and thereby reduce the incidence and severity of recurrent ocular disease was assessed in a mouse model. Mice latently infected with HSV-1 were vaccinated intranasally with a mixture of HSV-1 glycoproteins and recombinant Escherichia coli heat-labile enterotoxin B subunit (rEtxB) as an adjuvant. The systemic immune response induced was characterized by high levels of virus-specific immunoglobulin G1 (IgG1) in serum and very low levels of IgG2a. Mucosal immunity was demonstrated by high levels of IgA in eye and vaginal secretions. Proliferating T cells from lymph nodes of vaccinated animals produced higher levels of interleukin-10 (IL-10) than were produced by such cells from mock-vaccinated animals. This profile suggests that vaccination of latently infected mice modulates the Th1-dominated proinflammatory response usually induced upon infection. After reactivation of latent virus by UV irradiation, vaccinated mice showed reduced viral shedding in tears as well as a reduction in the incidence of recurrent herpetic corneal epithelial disease and stromal disease compared with mock-vaccinated mice. Moreover, vaccinated mice developing recurrent ocular disease showed less severe signs and a quicker recovery rate. Spread of virus to other areas close to the eye, such as the eyelid, was also significantly reduced. Encephalitis occurred in a small percentage (11%) of mock-vaccinated mice, but vaccinated animals were completely protected from such disease. The possible immune mechanisms involved in protection against recurrent ocular herpetic disease in therapeutically vaccinated animals are discussed.

Induction of mucosal immunity against herpes simplex virus type 1 in the mouse protects against ocular infection and establishment of latency.

Immune responses were assessed after intranasal immunization of mice with a mixture of herpes simplex virus type 1 (HSV-1) glycoproteins with cholera toxin and its B subunit as adjuvant. Antigen-specific serum antibodies, which were largely IgG with IgG1 the major subclass, neutralized virus in vitro with a titer equivalent to that elicited by active infection. Significant levels of antigen-specific IgA were found in mucosal fluids of the eye as well as the vagina. Lymphocytes from draining lymph nodes showed secondary proliferative responses when cultured with HSV-1 in vitro, in immunized mice only, with the production of interleukin-2, interferon-gamma, interleukin-4, and interleukin-5. After ocular challenge, immunized mice were protected against the development of severe eye disease, zosteriform spread, or encephalitis, whereas the incidence of clinical symptoms in mock-immunized mice was 83%, 74%, and 52%, respectively. Finally, the incidence of latency was reduced from 88% to 13% after intranasal immunization.

Survey of plastid RNA abundance during tomato fruit ripening: the amounts of RNA from the ORF 2280 region increase in chromoplasts.

A comprehensive survey of the levels of plastid RNAs at progressive stages of tomato fruit ripening was conducted by hybridizing total RNA with labeled Pst I fragments that cover almost the entire tomato plastid genome and with gene-specific probes. Two different cultivars of tomato (Lycopersicon esculentum Mill.) were examined, Traveler 76 and Count II. One of the tomato probes, P7, revealed a pronounced increase in the amount of an 8.3 kb RNA in ripe fruit. The homologous region of the tobacco plastid genome contains several genes for ribosomal proteins and a large unidentified open reading frame (2280 codons). Little change was observed in the levels of many transcripts during ripening. However, in some cases (e.g. psbA and psbC/D) the amount of RNA decreased during ripening of Count II but showed little or no change in Traveler 76. The contrast between Traveler 76 and Count II tomatoes shows that the level of plastid transcripts can vary substantially during fruit ripening with no obvious effect on the chloroplast to chromoplast transition. The large RNA from the P7 region may encode a protein that functions predominantly in chromoplasts.