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Compounds with nominally similar biological activity may exhibit differential toxicity due to differences in their interactions with cell membranes. Many pharmaceutical compounds are amphiphilic and can be taken up by phospholipid bilayers, interacting strongly with the lipid-aqueous interface whether or not subsequent permeation through the bilayer is possible. Bolaamphiphilic compounds, which possess two hydrophilic ends and a hydrophobic linker, can likewise undergo spontaneous uptake by bilayers. While membrane-spanning bolaamphiphiles can stabilize membranes, small molecules with this characteristic have the potential to create membrane defects via disruption of bilayer structure and dynamics. When compared to the amphiphilic therapeutic anticoagulant, warfarin, the bolaamphiphilic analogue, brodifacoum, exhibits heightened toxicity that goes beyond superior inhibition of the pharmacological target enzyme. We explore, herein, the consequences of anticoagulant accumulation in a dipalmitoylphosphatidylcholine (DPPC) bilayer. Coarse-grained molecular dynamics simulations reveal that permeation of phospholipid bilayers by brodifacoum causes a disruption of membrane barrier function that is driven by the bolaamphiphilic nature and size of this molecule. We find that brodifacoum partitioning into bilayers causes membrane thinning and permeabilization and promotes lipid flip-flop - phenomena that are suspected to play a role in triggering cell death. These phenomena are either absent or less pronounced in the case of the less toxic, amphiphilic compound, warfarin., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Nodular fasciitis (NF) is a relatively common mass-forming and self-limited subcutaneous pseudosarcomatous myofibroblastic proliferation of unknown pathogenesis. Due to its rapid growth and high mitotic activity, NF is often misdiagnosed as a sarcoma. While studying the USP6 biology in aneurysmal bone cyst and other mesenchymal tumors, we identified high expression levels of USP6 mRNA in two examples of NF. This finding led us to further examine the mechanisms underlying USP6 overexpression in these lesions. Upon subsequent investigation, genomic rearrangements of the USP6 locus were found in 92% (44 of 48) of NF. Rapid amplification of 5'-cDNA ends identified MYH9 as the translocation partner. RT-PCR and direct sequencing revealed the fusion of the MYH9 promoter region to the entire coding region of USP6. Control tumors and tissues were negative for this fusion. Xenografts of cells overexpressing USP6 in nude mice exhibited clinical and histological features similar to human NF. The identification of a sensitive and specific abnormality in NF holds the potential to be used diagnostically. Considering the self-limited nature of the lesion, NF may represent a model of 'transient neoplasia', as it is, to our knowledge, the first example of a self-limited human disease characterized by a recurrent somatic gene fusion event., (© 2011 USCAP, Inc All rights reserved)

The distinction between benign and malignant thyroid tumors in some cytological and histological specimens remains challenging. The aim of this study was to evaluate the use of High Mobility Group A2 (HMGA2) mRNA expression to distinguish benign from malignant thyroid tumors in cytological and histological specimens. RNA samples from 170 thyroid formalin-fixed paraffin-embedded (FFPE) tissues and 226 fine needle aspiration (FNA) specimens were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The FFPE tissues included 34 follicular adenomas, 10 Hürthle cell adenomas (HA), 6 hyperplastic nodules, 4 atypical adenomas, 44 classic papillary thyroid carcinomas (PTC), 29 follicular variant of PTC, 23 follicular thyroid carcinomas, 17 Hürthle cell carcinomas (HC), and 3 anaplastic thyroid carcinomas. The FNA specimens included 55 follicular adenomas, 34 HA, 20 hyperplastic nodules, 8 Hashimoto thyroiditis, 32 PTC, 24 follicular variant of PTC, 30 follicular thyroid carcinomas, 21 HC, and 2 anaplastic thyroid carcinomas. HMGA2 mRNA levels were expressed as relative fold change after normalizing with a calibrator. HMGA2 expression in thyroid carcinomas (16.8-fold for FFPE and 18.2-fold for FNA) was significantly higher than in benign lesions (0.8-fold for FFPE and 0.8-fold for FNA). HMGA2 expression in HC was relatively low (1.8-fold for FFPE and 8.5-fold for FNA) compared with the other types of carcinomas. HMGA2 expression values of 4.5-fold and 5.9-fold were used as cutoff points for FFPE and FNA (excluding HA and HC), respectively, to separate benign and malignant thyroid tumors, with 97.5% clinical specificity and 79.8% sensitivity for FFPE, and 95.2% clinical specificity and 88.6% sensitivity for the FNA specimens. Conventional RT-PCR supported the qRT-PCR results. Detection of HMGA2 mRNA expression by qRT-PCR may be a useful tool to assist in the diagnosis of well-differentiated thyroid carcinomas. The 1-step qRT-PCR method is a sensitive, accurate, and reliable technique for gene expression analysis of thyroid tumors.

The capability of molecular markers to differentiate between benign and malignant well-differentiated thyroid tumors remains unclear. The aim of this study was to evaluate the use of insulin-like growth factor II mRNA binding protein-3 (IMP3) mRNA expression to distinguish benign from malignant thyroid tumors. RNA samples from 80 formalin-fixed, paraffin-embedded thyroid tissues, including 22 usual papillary thyroid carcinomas (PTCs), 18 follicular variants of PTC, 5 follicular thyroid carcinomas, 33 follicular adenomas, and 2 hyperplastic nodules, were used for quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. IMP3 mRNA expression levels in thyroid tumors were expressed as relative fold change (fold) after normalization with normal thyroid RNA. The results showed that thyroid carcinomas including PTC, follicular variants of PTC, and follicular thyroid carcinomas have significantly higher IMP3 expression levels with 48.3, 35.3, and 43.8 fold, respectively, compared with benign thyroid lesions (2.8 fold). Using the IMP3 expression value of 5 fold as a cutoff point to separate benign and malignant thyroid tumors, IMP3 qRT-PCR analysis had a 91.4% clinical specificity and 86.7% clinical sensitivity for the diagnosis of well-differentiated thyroid carcinomas. Conventional RT-PCR and immunohistochemical analysis for IMP3 in a subset of cases supported the qRT-PCR results. These results indicate that detection of IMP3 mRNA expression levels by qRT-PCR may be a useful molecular marker to assist in the diagnosis of well-differentiated thyroid carcinomas.

The discrimination between well-differentiated liposarcomas/atypical lipomatous tumors and lipomas can be diagnostically challenging at the histological level. However, cytogenetic identification of ring and giant rod chromosomes supports the diagnosis of well-differentiated liposarcoma/atypical lipomatous tumor. These abnormal chromosomes are mainly composed of amplified genomic sequences derived from chromosome 12q13-15, and contain several genes, including MDM2, CDK4 (SAS), TSPAN31, HMGA2, and others. MDM2 is consistently amplified in well-differentiated liposarcomas/atypical lipomatous tumors, and up to 25% in other sarcomas. As part of a large genomic study of lipomatous neoplasms, we initially found CPM to be consistently amplified in well-differentiated liposarcomas/atypical lipomatous tumors. To further explore this initial finding, we investigated the copy number status of MDM2 and CPM by fluorescent in situ hybridization (FISH) on a series of 138 tumors and 17 normal tissues, including 32 well-differentiated liposarcoma/atypical lipomatous tumors, 63 lipomas, 11 pleomorphic lipomas, 2 lipoblastomas, 30 other tumors and 17 normal fat samples. All 32 well-differentiated liposarcoma/atypical lipomatous tumors showed amplification of MDM2 and CPM, usually >20 copies per cell. The other tumors lacked MDM2 and/or CPM amplification. Chromogenic in situ hybridization confirmed the above results on a subset of these tumors (n=27). These findings suggest that identification of CPM amplification could be used as an alternative diagnostic tool for the diagnosis of well-differentiated liposarcoma/atypical lipomatous tumors.

We present a draft sequence of the genome of Aedes aegypti, the primary vector for yellow fever and dengue fever, which at approximately 1376 million base pairs is about 5 times the size of the genome of the malaria vector Anopheles gambiae. Nearly 50% of the Ae. aegypti genome consists of transposable elements. These contribute to a factor of approximately 4 to 6 increase in average gene length and in sizes of intergenic regions relative to An. gambiae and Drosophila melanogaster. Nonetheless, chromosomal synteny is generally maintained among all three insects, although conservation of orthologous gene order is higher (by a factor of approximately 2) between the mosquito species than between either of them and the fruit fly. An increase in genes encoding odorant binding, cytochrome P450, and cuticle domains relative to An. gambiae suggests that members of these protein families underpin some of the biological differences between the two mosquito species.

Invasion of the vector salivary glands by Plasmodium is a critical step for malaria transmission. To describe salivary gland cellular responses to sporozoite invasion, we have undertaken the analysis of Anopheles gambiae salivary gland transcriptome using Serial Analysis of Gene Expression (SAGE). Statistical analysis of the more than 160000 sequenced tags generated from four libraries, two from glands infected by Plasmodium berghei, two from glands of controls, revealed that at least 57 Anopheles genes are differentially expressed in infected salivary glands. Among the 37 immune-related genes identified by SAGE tags, four (Defensin1, GNBP, Serpin6 and Cecropin2) were found to be upregulated during salivary gland invasion, while five genes encoding small secreted proteins display induction patterns strongly reminiscent of that of Cecropin2. Invasion by Plasmodium has also an impact on the expression of genes involved in transport, lipid and energy metabolism, suggesting that the sporozoite may exploit the metabolism of its host. In contrast, protein composition of saliva is predicted to be only slightly modified after infection. This study, which is the first transcriptome analysis of the salivary gland response to Plasmodium infection, provides a basis for a better understanding of Plasmodium/Anopheles salivary gland interactions.

Plasmodium, the malaria parasite, undergoes a complex developmental program in its mosquito vector. The ookinete is the parasite form which invades the mosquito midgut and is an important stage for genetic mixing. To identify genes expressed during ookinete development and mosquito midgut invasion, purified zygotes and ookinetes of the rodent parasite Plasmodium berghei were used to construct a suppression subtractive hybridization cDNA library, enriched in sequences expressed in the ookinete stage. In addition to four genes coding for previously described major ookinete-secreted proteins, we isolated ookinete-expressed sequences representing 18 predicted genes. Their gene products include proteins involved in signal transduction and regulatory processes. For six of these genes our analysis provides the first evidence for expression in the ookinete stage. A majority of the genes are not expressed in the zygote, the preceding developmental stage. Furthermore, four of the genes are also transcribed in sporozoites, and one of these in merozoites, suggesting that they code for proteins with a function common to Plasmodium invasive stages.

The only natural mechanism of malaria transmission in sub-Saharan Africa is the mosquito, generally Anopheles gambiae. Blocking malaria parasite transmission by stopping the development of Plasmodium in the insect vector would provide a useful alternative to the current methods of malaria control. Toward this end, it is important to understand the molecular basis of the malaria parasite refractory phenotype in An. gambiae mosquito strains. We have selected and sequenced six bacterial artificial chromosome (BAC) clones from the Pen-1 region that is the major quantitative trait locus involved in Plasmodium encapsulation. The sequence and the annotation of five overlapping BAC clones plus one adjacent, but not contiguous clone, totaling 585kb of genomic sequence from the centromeric end of the Pen-1 region of the PEST strain were compared to that of the genome sequence of the same strain produced by the whole genome shotgun technique. This project identified 23 putative mosquito genes plus putative copies of the retrotransposable elements BEL12 and TRANSIBN1_AG in the six BAC clones. Nineteen of the predicted genes are most similar to their Drosophila melanogaster homologs while one is more closely related to vertebrate genes. Comparison of these new BAC sequences plus previously published BAC sequences to the cognate region of the assembled genome sequence identified three retrotransposons present in one sequence version but not the other. One of these elements, Indy, has not been previously described. These observations provide evidence for the recent active transposition of these elements and demonstrate the plasticity of the Anopheles genome. The BAC sequences strongly support the public whole genome shotgun assembly and automatic annotation while also demonstrating the benefit of complementary genome sequences and of human curation. Importantly, the data demonstrate the differences in the genome sequence of an individual mosquito compared to that of a hypothetical, average genome sequence generated by whole genome shotgun assembly.

We describe the preliminary analysis of over 35,000 clones from a full-length enriched cDNA library from the malaria mosquito vector Anopheles gambiae. The clones define nearly 3,700 genes, of which around 2,600 significantly improve current gene definitions. An additional 17% of the genes were not previously annotated, suggesting that an equal percentage may be missing from the current Anopheles genome annotation.

We performed genome-wide sequence comparisons at the protein coding level between the genome sequences of Drosophila melanogaster and Anopheles gambiae. Such comparisons detect evolutionarily conserved regions (ecores) that can be used for a qualitative and quantitative evaluation of the available annotations of both genomes. They also provide novel candidate features for annotation. The percentage of ecores mapping outside annotations in the A. gambiae genome is about fourfold higher than in D. melanogaster. The A. gambiae genome assembly also contains a high proportion of duplicated ecores, possibly resulting from artefactual sequence duplications in the genome assembly. The occurrence of 4063 ecores in the D. melanogaster genome outside annotations suggests that some genes are not yet or only partially annotated. The present work illustrates the power of comparative genomics approaches towards an exhaustive and accurate establishment of gene models and gene catalogues in insect genomes.

The Anopheles gambiae genome sequence has been analyzed to find ATP-binding cassette protein genes based on deduced protein similarity to known family members. A nonredundant collection of 44 putative genes was identified including five genes not detected by the original Anopheles genome project machine annotation. These genes encode at least one member of all the human and Drosophila melanogaster ATP-binding protein subgroups. Like D. melanogaster, A. gambiae has subgroup ABCH genes encoding proteins different from the ABC proteins found in other complex organisms. The largest Anopheles subgroup is the ABCC genes which includes one member that can potentially encode ten different isoforms of the protein by differential splicing. As with Drosophila, the second largest Anopheles group is the ABCG subgroup with 12 genes compared to 15 genes in D. melanogaster, but only 5 genes in the human genome. In contrast, fewer ABCA and ABCB genes were identified in the mosquito genome than in the human or Drosophila genomes. Gene duplication is very evident in the Anopheles ABC genes with two groups of four genes, one group with three genes and three groups with two head to tail duplicated genes. These characteristics argue that the A. gambiae is actively using gene duplication as a mechanism to drive genetic variation in this important gene group.

Drosophila melanogaster has a gene very similar to human MRP1 that encodes a full ABC-transporter containing three membrane-spanning domains and two nucleotide-binding domains. This 19 exon insect gene, dMRP (FBgn0032456), spans slightly more than 22 kb. The cDNA SD07655 representing this gene was sequenced and found to contain sequences from 12 exons including single copies of two exons having multiple genomic copies. The gene contains two variant copies of exon 4 and seven of exon 8. While a cDNA contains only one version of each variable exon, all forms of these variable exons were detected in adult fly mRNA. These results predict that Drosophila could make 14 different MRP isoforms from a single gene by substituting different variable exons. This is the first report of any organism using differential splicing of alternative, internal exons, to produce such a large array of MRP isoforms having the same size, but with limited and defined internal variations. Defining the functional differences in the dMRP isoforms should provide clues to the structure/function relationships of the amino acids in these MRP domains, both for the insect enzyme and for those of other species.

Anopheles gambiae is the principal vector of malaria, a disease that afflicts more than 500 million people and causes more than 1 million deaths each year. Tenfold shotgun sequence coverage was obtained from the PEST strain of A. gambiae and assembled into scaffolds that span 278 million base pairs. A total of 91% of the genome was organized in 303 scaffolds; the largest scaffold was 23.1 million base pairs. There was substantial genetic variation within this strain, and the apparent existence of two haplotypes of approximately equal frequency ("dual haplotypes") in a substantial fraction of the genome likely reflects the outbred nature of the PEST strain. The sequence produced a conservative inference of more than 400,000 single-nucleotide polymorphisms that showed a markedly bimodal density distribution. Analysis of the genome sequence revealed strong evidence for about 14,000 protein-encoding transcripts. Prominent expansions in specific families of proteins likely involved in cell adhesion and immunity were noted. An expressed sequence tag analysis of genes regulated by blood feeding provided insights into the physiological adaptations of a hematophagous insect.

Cytochrome P450s are a superfamily of haemoproteins, important in the metabolism of endogenous compounds and xenobiotics. As a first step to elucidating the role of this family in insecticide resistance in the malaria mosquito, Anopheles gambiae, we have cloned and mapped multiple P450 genes. Sixteen cDNAs encoding full-length P450s were cloned and physically mapped to the mosquito's polytene chromosomes. Fourteen of these encode putative CYP6 proteins and two encode P450s belonging to the CYP9 class. Eighteen new A. gambiae Cyp4 P450 genes were identified using degenerate PCR primers, cDNAs were detected for ten and in situ locations for thirteen members of this gene family.

The sequence and cytological location of five Anopheles gambiae glutathione S-transferase (GST) genes are described. Three of these genes, aggst1-8, aggst1-9 and aggst1-10, belong to the insect class I family and are located on chromosome 2R, in close proximity to previously described members of this gene family. The remaining two genes, aggst3-1 and aggst3-2, have a low sequence similarity to either of the two previously recognized classes of insect GSTs and this prompted a re-evaluation of the classification of insect GST enzymes. We provide evidence for seven possible classes of insect protein with GST-like subunits. Four of these contain sequences with significant similarities to mammalian GSTs. The largest novel insect GST class, class III, contains functional GST enzymes including two of the A. gambiae GSTs described in this report and GSTs from Drosophila melanogaster, Musca domestica, Manduca sexta and Plutella xylostella. The genes encoding the class III GST of A. gambiae map to a region of the genome on chromosome 3R that contains a major DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] resistance gene, suggesting that this gene family is involved in GST-based resistance in this important malaria vector. In further support of their role in resistance, we show that the mRNA levels of aggst3-2 are approx. 5-fold higher in a DDT resistant strain than in the susceptible strain and demonstrate that recombinant AgGST3-2 has very high DDT dehydrochlorinase activity.

Background: Leishmaniases are major parasitic diseases caused by protozoans that are obligate intracellular parasites during the mammalian phase of their life cycle. Quantitation of experimental mammalian cell infections is usually performed by time-consuming microscopic examination. In this report a flow cytometry (FCM)-based assay suitable for studying in vitro infections by L.amazonensis is presented., Methods: Intense fluorescence staining of the amastigote forms with a stage- and species-specific monoclonal antibody was obtained after permeabilization of both the host-cell cytoplasmic membrane and the parasitophorous vacuole membrane by saponin treatment., Results: Upon flow cytometry (FCM) analysis, parasitized cells separated sharply from the auto-fluorescence of the mammalian host cells, giving the assay a high degree of sensitivity and specificity. Ninety to 98% of cells in the more fluorescent population harbored parasites visible by phase-contrast and UV-light microscopy, while no parasites were observed in more than 95% of the cells in the population with background fluorescence. Comparisons of the FCM results with those from microscope counting and analysis of various dilutions of parasitized cells confirmed the reliability of the method., Conclusions: The FCM assay provided rapid quantitation of Leishmania infection either in mouse macrophages, the natural host cell in murine leishmaniasis, or in Chinese hamster ovary (CHO) cells, a non-macrophage cell line proposed as an in vitro model for studying host-parasite interactions. The protocol described here should be adaptable to studies involving other parasites residing in nucleated cells., (Copyright 2000 Wiley-Liss, Inc.)

Tyrosine kinase sequences were identified and characterized in Anopheles gambiae, the major vector of malaria in subsaharan Africa. One of these sequences has the characteristics expected for a homologue of the Drosophila sevenless gene, which is necessary for R7 photoreceptor cell fate determination in the developing compound eye. The putative Anopheles sevenless gene homologue is located in a telomeric region of the X chromosome and is expressed in the head of late larval and pupal stage mosquitoes. Identification of the Anopheles homologue of the sevenless gene is a first step towards the development of a dominant phenotypic marker that could be used for detecting transformed Anopheles mosquitoes in a wide variety of genetic backgrounds and, as such, could be used in the development of transgenic mosquitoes for the control of parasite transmission. Preliminary evidence for sevenless sequences were also found in DNA from blackfly, Mediterranean fruit fly and the honeybee.

The molecular structure of the telomeric region at the left arm of the second chromosome of the mosquito Anopheles gambiae has been determined in the transformed strain G418 that contains a pUChsneo transgene attached at the 2L chromosome end, and in the Pink eye laboratory strain (PE). Both strains contain the same complex satellite positioned distal to a unique region. FIGE mapping of the telomeric region of the PE strain revealed distinct DNA fragment lengths that segregated with individual chromosomes. Genomic DNA fragments were cloned from the 2L telomeric region, which accounted for about half of 2L chromosomes in the PE population. In all three cases studied, long fragments of different middle repetitive sequences were found attached to the distal ends of the 2L satellite. We propose that random fragments of DNA may be occasionally added during recombination between complex satellite repeats at the chromosome ends.

One of the functions of telomeres is to counteract the terminal nucleotide loss associated with DNA replication. While the vast majority of eukaryotic organisms maintain their chromosome ends via telomerase, an enzyme system that generates short, tandem repeats on the ends of chromosomes, other mechanisms such as the transposition of retrotransposons or recombination can also be used in some species. Chromosome end regression and extension were studied in a medically important mosquito, the malaria vector Anopheles gambiae, to determine how this dipteran insect maintains its chromosome ends. The insertion of a transgenic pUChsneo plasmid at the left end of chromosome 2 provided a unique marker for measuring the dynamics of the 2L telomere over a period of about 3 years. The terminal length was relatively uniform in the 1993 population with the chromosomes ending within the white gene sequence of the inserted transgene. Cloned terminal chromosome fragments did not end in short repeat sequences that could have been synthesized by telomerase. By late 1995, the chromosome ends had become heterogeneous: some had further shortened while other chromosomes had been elongated by regenerating part of the integrated pUChsneo plasmid. A model is presented for extension of the 2L chromosome by recombination between homologous 2L chromosome ends by using the partial plasmid duplication generated during its original integration. It is postulated that this mechanism is also important in wild-type telomere elongation.

African trypanosomes are covered by a dense protein layer that is immunologically distinct on different trypanosome isolates and is termed the variant surface glycoprotein (VSG). The different VSGs are expressed in a general order, where some VSGs appear preferentially early in infection and others only later. The exposed epitopes on a late antigen, VSG 78, of T.equiperdum were studied by the technique of monoclonal antibody (MAb) escape selection. MAbs that neutralize trypanosomes bearing VSG 78 reacted with the VSG only when it was attached to the trypanosome surface, suggesting that the most immunogenic surface epitopes are conformational. Trypanosome clones resistant to one of the MAbs yet still expressing VSG 78 or 78(20) were isolated in vitro. Two independent variants resistant to MAb H3 changed Ser192 to Arg by a single base change in the VSG gene and a variant resistant to MAb H21 had a single base change that converted Gln172 to Glu. A variant resistant to MAb H7 had several changes in the VSG gene, a gene conversion in the 5' region and an isolated mutation in codon 220 that is proposed to be responsible for the resistance phenotype. The isotypic bias of the MAbs against VSG 78 and an analysis of the natural variants that are resistant to MAb 78H21 suggest that glycosylation plays a role in the immunogenicity of these proteins. The analysis defines some of the exposed amino acid residues and demonstrates that VSG genes are altered by mutations and small gene conversions as well as replaced by large gene conversion-like events. The results provide biological data supporting the model of VSG structure obtained by crystallographic studies.

A nontransformed line of Chinese hamster ovary (CHO) cells (Pollard and Stanners, 1979) has been transformed by the Schmidt-Ruppin subgroup D strain of Rous sarcoma virus (SR-RSV). SR-RSV transformed CHO cells are shown to differ from spontaneously transformed cells in that the virally transformed cells are more resistant to growth inhibition or changes in cell shape by 8-Br-cyclic AMP or cholera toxin. SR-RSV transformed rat (NRK) cells also have a reduced sensitivity to growth inhibition by 8-Br-cyclic AMP. Cyclic AMP-dependent protein kinase was examined in SR-RSV transformed CHO cells, but no differences in enzyme level, activation by cyclic AMP, chromatographic behavior, or its ability to phosphorylate endogenous proteins in whole cells could be detected. It is concluded that transformation of CHO and NRK cells by SR-RSV alters the cells in a manner different from spontaneous transformation, and that this alteration does not affect cAMP-dependent protein kinase activity.

The expression of Trypanosoma equiperdum variant surface protein (VSG) 78 is accomplished by the duplicative transposition of silent basic copy (BC) genes into a telomer-linked expression site to form an expression-linked copy (ELC). In two independent isolates expressing VSG 78, the ELC is a composite gene. The analysis of VSG 78 cDNA clones from these two Bo Tat 78 isolates and the respective BC genes revealed that both ELCs were constructed from the same three BC genes, a 3' BC which donated the last 255 bp of each ELC and two closely related 5' BCs. Although sequences of both 5' BC genes were found in each ELC, the junction with the 3' BC was provided by the same 5' BC in both cases. This 5' BC is an incomplete gene with insufficient open reading frame to code for a complete VSG and thus can only be used when joined to a competent 3' end. Furthermore, both 5' BC genes lack a conserved 14 nucleotide sequence found on all VSG mRNAs. These results support a model in which composite gene formation plays a role in the determination of the order of VSG expression. They also illustrate similarities between immunoglobulin gene and VSG gene construction.

Nuclear magnetic resonance (NMR) spectroscopy was used to quantitate abundance of 2H in body water of human infants. This method provides precise measurement of total-body water without the extensive sample preparation requirements of previously described methods for determination of 2H content in body fluids. 2H2O (1 g/kg body weight) was administered to infants and saliva and urine were collected for up to 5 h. An internal standard was added directly to the fluid specimen and 2H enrichment in water was measured by NMR spectroscopy. Working range of deuterium abundance was 0.04-0.32 atom %. Coefficients of variation for saliva samples at 0.20 atom % 2H was 1.97%. 2H content in urine and saliva water reached a plateau by 4 h after administration, and amounts in the two fluids were virtually identical. Mean total-body water determination for six infants was 58.3 +/- 5.8% of body weight (range 53-66%).

Treatment of growing Rous sarcoma virus-transformed Chinese hamster ovary cells with the cyclic AMP analog 8-bromo-cyclic adenosine 3',5'-monophosphate (8-bromo-cyclic AMP) stimulates the incorporation of 32Pi into the viral transforming protein pp60src. Based on one-dimensional and two-dimensional peptide analysis and phosphoamino acid analysis, the increase is on a single phosphoserine residue at the NH2 terminus of the protein. The phosphate incorporation increases during the first 4 h of treatment. The pp60src kinase activity in extracts of cells treated with 8-bromo-cyclic AMP was stimulated about 2- to 3-fold. This stimulation of kinase activity increased during the first 3 h of treatment with 1 mM 8-bromo-cAMP and the activity was increased in both the soluble and particulate fraction of the cells. These results suggest that cyclic AMP can modulate the activity of pp60src in transformed cells.

5-Methyltryptophan-resistant mutants derived from Bacillus subtilis strain 168 synthesize all of the tryptophan biosynthetic enzymes constitutively and excrete tryptophan. These mutants can be divided into three classes: class 1, low enzyme level and low rate of tryptophan excretion; class 2, high enzyme level and intermediate rate of tryptophan excretion; and class 3, high enzyme level and high rate of tryptophan excretion. A bradytrophic requirement for phenylalanine is correlated with the rate of tryptophan excretion. The phenylalanine requirement is relieved when the rate of tryptophan excretion is reduced by either (i) lowering the level of the tryptophan enzymes, (ii) reducing the supply of a tryptophan precursor (chorismate), or (iii) stopping tryptophan synthesis by a mutational block in the pathway. All of the mutants map in a region of the chromosome previously reported as the mtr locus. Our data show that synthesis of the tryptophan enzymes is controlled through the mtr locus but not influenced by precursors of tryptophan.

Bacillus alvei excretes indole during early exponential growth in acid-hydrolyzed casein medium. l-Threonine is the amino acid responsible for "early" indole excretion, and the amount of indole excreted is directly related to the amount of l-threonine in the medium. "Early-indole" excretion can be prevented by the continuous addition of serine (3.1 mumoles per ml per hr) or by substituting a mutant with an impaired ability to degrade serine. The addition of serine to a culture during the period of indole excretion halts the excretion and stimulates indole utilization. Threonine is a competitive inhibitor of serine (K(i) = 0.6 m) in the tryptophan synthetase B reaction. The internal tryptophan concentration increases during the period of indole excretion, suggesting that threonine acts by increasing the activity of the tryptophan pathway. This view is supported by experiments demonstrating that anthranilic acid and indoleacrylic acid also stimulate indole excretion. A metabolic explanation is offered and discussed.

Nicotinamide adenine dinucleotide phosphate (reduced form) is formed more rapidly after the addition of thiosulfate to suspensions of intact Thiobacillus neapolitanus in the absence of CO(2) than nicotinamide adenine dinucleotide (reduced form). Measurement of acid-stable metabolites shows this phenomenon to be the result of rapid reoxidation of nicotinamide adenine dinucleotide (reduced form) by 3-phosphoglyceric acid and other oxidized intermediates, which are converted to triose and hexose phosphates, and that, in reality, the rate of nicotinamide adenine dinucleotide (oxidized form) reduction exceeds that of nicotinamide adenine dinucleotide phosphate (oxidized form) by approximately 4.5-fold. The overall rate of pyridine nucleotide reduction by thiosulfate (264 nmol per min per mg of protein) is in excess of that rate needed to sustain growth. Pyridine nucleotide reduction, adenosine triphosphate synthesis, and carbohydrate synthesis are prevented by the uncoupler m-Cl-Carbonylcyanide phenylhydrazone. Sodium amytal inhibits pyridine nucleotide reduction and carbohydrate synthesis are prevented by the uncoupler m-Cl-carbonylcyanide observations are reproduced when sulfide serves as the substrate. The rate of pyridine nucleotide anaerobic reduction with endogenous substrates or thiosulfate is less than 1% of the aerobic rate with thiosulfate. We conclude that the principal, if not the only, pathway of pyridine nucleotide reduction proceeds through an energy-dependent and amytal-sensitive step when either thiosulfate or sulfide is used as the substrate.