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Obstructive sleep-disordered breathing is common in children. From 3 percent to 12 percent of children snore, while obstructive sleep apnea syndrome affects 1 percent to 10 percent of children. The majority of these children have mild symptoms, and many outgrow the condition. Consequences of untreated obstructive sleep apnea include failure to thrive, enuresis, attention-deficit disorder, behavior problems, poor academic performance, and cardiopulmonary disease. The most common etiology of obstructive sleep apnea is adenotonsillar hypertrophy. Clinical diagnosis of obstructive sleep apnea is reliable; however, the gold standard evaluation is overnight polysomnography. Treatment includes the use of continuous positive airway pressure and weight loss in obese children. These alternatives are tolerated poorly in children and rarely are considered primary therapy. Adenotonsillectomy is curative in most patients. Children with craniofacial syndromes, neuromuscular diseases, medical comorbidities, or severe obstructive sleep apnea, and those younger than three years are at increased risk of developing postoperative complications and should be monitored overnight in the hospital. [ABSTRACT FROM AUTHOR]

The academic successes of AM during the past 2 decades are marked by board certification, fellowship program accreditation, residency curricula creation, and the evolution of a remarkably respected scientific journal, the Journal of Adolescent Health. These same accomplishments have increased professional and public recognition of unmet population needs and the specialists who can help address them. The adolescent population is large, diverse, underserved, and characterized by increasingly complex medical and behavioral issues. Meeting their health care needs is a national priority. Primary care professionals who treat adolescents want and need adolescent-specific training in anticipatory guidance, screening, counseling, and management of common adolescent problems. A larger workforce of AM physicians is needed to provide this training, consult on complex medical and psychosocial issues when requested, and lead research efforts that will advance knowledge in the field. Developing this workforce will require improved recruitment into fellowship training; mentorship, policies, and resources that support trainee and faculty diversity; and articulation of the skills that define an AM physician.

Using the 2001-2004 Medical Expenditures Panel Survey, we examined rates of past-year adolescent time alone with a clinician by visit type, and among youths with a preventive visit, examined age, gender, and race/ethnicity differences. Youths with a preventive visit have higher rates of time alone; rates for these youths increase with age, are higher for males (42%) versus females (37%), and are lowest among Hispanics. Time alone rates are low, especially for younger females and Hispanic youths. Special efforts are needed to increase time alone in these populations., (Copyright 2010 Society for Adolescent Medicine. Published by Elsevier Inc. All rights reserved.)

Down syndrome, Turner syndrome, and Klinefelter syndrome constitute the most common chromosomal abnormalities encountered by primary care physicians. Down syndrome typically is recognized at birth, Turner syndrome often is not recognized until adolescence,and many men with Klinefelter syndrome are never diagnosed. Although each syndrome is caused by an abnormal number of chromosomes, or aneuploidy, they are distinct syndromes with learning disabilities and a predisposition toward autoimmune diseases,endocrinologic disorders, and cancers. Optimal health care requires a thorough knowledge of the unique health risks, psychoeducational needs, functional capabilities, and phenotypic variation associated with each condition. Syndrome-specific health care should complement standard preventive health care recommendations. Checklists and syndrome-specific growth grids should be used. Ongoing communication between specialists and primary care physicians and between pediatric and adult clinicians is essential. Support groups and Internet resources can benefit affected individuals and their families immensely.

A unique family of genes encoding serine endoproteases related to the Saccharomyces cerevisiae serine endoprotease kexin was identified in Pneumocystis carinii. Unlike previously described serine endoprotease genes that are single copies, multiple copies of the P. carinii serine endoprotease are distributed throughout the genome. The proteins predicted by these variant genes demonstrate sequence variability, but they retain the conserved active sites associated with endoprotease activity. The serine endoprotease was localized to the organism surface by immunohistochemical and immunofluorescence studies and to the electron lucent layer of the cyst wall by immunoelectron microscopy. The findings of multiple copies of the serine endoprotease gene in the P. carinii genome, and its localization to the cell surface, suggest that this protease plays an important role in the biology of P. carinii and that antigenic variation of the surface-expressed serine endoprotease may be a strategy for immune evasion. P. carinii serine endoprotease provides a novel target for chemotherapeutic and immune-based approaches to the treatment of P. carinii pneumonia.

Cryptococcus neoformans possesses two mating types, MATalpha and MATa. Alpha-Cells are more virulent than a-cells and are also, unlike a-cells, capable of producing extensive hyphae in the haploid phase. The molecular analysis of hyphae production in C. neoformans has resulted in the identification of a gene which displays substantial similarity to other fungal STE12 genes, including the presence of a highly conserved homeodomain. Overexpression of the C. neoformans gene resulted in poor growth, altered morphology and the presence of hyphal projections, phenotypes reported in similar studies of the Saccharomyces cerevisiae STE12 gene. Overexpression was also found to induce MFalpha, a pheromone, and CNLAC1, a confirmed C. neoformans virulence gene. The C. neoformans STE12alpha gene, however, has one striking difference from other fungal STE12 genes; it is found only in alpha-cells. The existence of STE12alpha in C. neoformans suggests that this fungus has elements of a conserved MAP kinase cascade, which may be organized in a novel manner.

The major surface glycoprotein (MSG) of Pneumocystis carinii is encoded by a family of related but distinct genes distributed throughout the P. carinii genome. Previous reports of the genomic and mRNA MSG structure suggested that there was a highly conserved 5'-untranslated region and a highly variable translated region. In the current study, we demonstrate that there is a single expression site for MSG expression and that different MSG genes are located downstream of this expression site. Isolation of a genomic clone containing the putative 5'-untranslated region has demonstrated that there was a single base sequencing error in what was considered to be the untranslated region. The corrected sequence reveals an extended open reading frame encoding a constant amino-terminal leader domain, with a typical signal peptide, for the MSG protein family. Since this constant amino-terminal domain is encoded by a single copy genomic sequence, a recombination/gene conversion-mediated antigenic switching event is required to effect the known variability in expressed MSG sequences. Therefore, like some bacterial and protozoan pathogens, the opportunistic fungal pathogen P. carinii contains a constant genomic site dedicated to MSG expression and a switchable downstream region for the variable part of the MSG gene family.

Cryptococcus neoformans is a major opportunistic fungal pathogen in AIDS and other immunosuppressed patients. We have shown that wild-type haploid C. neoformans can develop an extensive hyphal phase under appropriate conditions. Hyphae produced under these conditions are monokaryotic, possess unfused clamp connections, and develop basidia with viable basidiospores. The ability to undergo this transition is determined by the presence of the alpha-mating type locus and is independent of serotype. The association of the hyphal phase with the alpha-mating type may explain the preponderance of this mating type in the environment and the nature of the infectious propagule of C. neoformans.

A Cryptococcus neoformans galactose auxotroph was created by ultraviolet light mutagenesis and complemented with a C. neoformans genomic library. The translated sequence of the complementing DNA revealed a high degree of similarity to a number of UDP glucose-D-galactose-1-phosphate uridylyltransferases. Expression of C. neoformans GAL7 mRNA followed a pattern similar to Saccharomyces cerevisiae expression; it was first observed within 2.5 min of induction and fully induced by 30 min. The gene was completely repressed in the presence of glucose. The GAL7 promoter was isolated and used to construct a promoter cassette. Two genes were tested in this cassette for galactose regulation by creating GAL7 promoter fusions with their coding regions. MF alpha, which encodes a pheromone, was found to produce filaments only in transformants that were induced by galactose. A second gene, beta-glucuronidase (gusA), which is a commonly used reporter gene, was tested and also found to be expressed. When the GAL7p::GUS fusion was used to quantify inducibility of the GAL7 promoter, the level of enzyme activity was at least 500-fold greater for cells grown in galactose than for cells grown in glucose. The GAL7 promoter is the first inducible promoter characterized in C. neoformans and the GUS gene is the first heterologous gene shown to be expressed in this yeast pathogen.

An initial and crucial step in the establishment of many microbial infections is the attachment of the pathogen to the host cells. Thus, adherence of Pneumocystis carinii (Pc) to type I pneumocytes is believed to be important in the induction of Pc pneumonia. Little is known about the nature of the attachment of Pc to type I cells, although extracellular matrix (ECM) proteins, such as fibronectin and laminin, have been implicated in the process. We report here the isolation of a Pc gene encoding a receptor protein that binds both fibronectin and laminin in vitro. A cDNA clone encoding the Pc ECM receptor was isolated from a Pc cDNA library and identified on the basis of sequence homology to the human colon carcinoma laminin receptor. Southern blot analysis of Pc genomic DNA confirmed that the cDNA was of Pc origin. Northern blot analysis of Pc total RNA showed a predominant mRNA of approximately 1400 nucleotides that hybridized to the ECM receptor gene. The ECM receptor predicted from the cDNA sequence is 295 amino acid residues long, with a molecular mass of 32.8 kDa. The C-terminal third of the polypeptide is highly negatively charged, whereas the N-terminal two-thirds contains hydrophobic segments that may play a role in membrane association. Sequence analysis and alignment of the N terminus with the laminin receptor cDNA sequence of human colon carcinoma support the conclusion that the Pc ECM receptor cDNA clone is a full-length clone. A Western blot of the overexpressed ECM receptor protein bound both laminin and fibronectin in vitro. Antibodies raised to the overexpressed receptor protein interacted with a 33-kDa protein in total Pc cell lysates. These findings raise the possibility that the Pc ECM receptor protein may mediate the organism's attachment to type I pneumocytes and, thus, may play a crucial role in Pc pathogenesis.

A cDNA from Cryptococcus neoformans, encoding imidazole glycerol phosphate dehydratase (IGPD), was isolated by complementation of a his3 mutant strain of Saccharomyces cerevisiae. The C. neoformans HIS3 cDNA encodes an approx. 22-kDa protein with a high degree of amino-acid sequence similarity to IGPDs from ten other microorganisms, as well as Arabidopsis thaliana. Most striking are two conserved HHXXE regions and several conserved His, Asp and Glu residues. The cDNA was engineered for expression in Escherichia coli and an approx. 26-kDa protein was identified by SDS-PAGE. DNA and N-terminal sequence analyses confirmed that this protein was C. neoformans IGPD. Furthermore, IGPD assays of crude extracts from IGPD-producing E. coli cells demonstrated that the C. neoformans protein was catalytically active.

Cryptococcus neoformans is a significant fungal pathogen in immunocompromised patients. The ability of C. neoformans to produce melanin has been correlated with virulence. The role of melanin in promoting virulence is unclear, although an anti-oxidant function has been suggested. To begin to define the genetic mechanisms responsible for melanin production in C. neoformans, we describe the isolation of seven melanin-deficient mutant classes. Some of the mutants can be suppressed by addition of Cu2+ to media, suggesting that the phenoloxidase of C. neoformans, like other fungal phenoloxidases, contains copper. Other mutants display a recessive sterile phenotype. A genetic and phenotypic characterisation of these mutants is presented.

The Saccharomyces cerevisiae gene for para-aminobenzoate (PABA) synthase has been identified based upon its ability to confer sulfonamide resistance when present on a multicopy episomal vector. The 3840 bp DNA sequence fragment reported here contains a 2199 bp open reading frame encoding a 733 amino acid protein with similarity to the two components of PABA synthase described for prokaryotes (Escherichia coli PabA and PabB), suggesting that PABA synthase is bifunctional in yeast. The cloned sequence was confirmed to be PABA synthase by gene disruption. Chromosome gel analysis places the gene for PABA synthase on chromosome XIV.

The Cryptococcus neoformans dihydrofolate reductase (DHFR) gene has been isolated from cDNA and genomic DNA libraries. The 690-base pair coding sequence codes for a 25,152-Da protein, which is the largest monofunctional DHFR yet reported. The gene contains two introns, and several putative regulatory sequences have been identified. The coding sequence was placed in a pUC-based expression vector, which expresses C. neoformans DHFR in Escherichia coli at a level of about 5% of the total soluble extract. The expressed DHFR was purified to homogeneity by methotrexate-Sepharose affinity chromatography, followed by anion exchange chromatography on Q-Sepharose. On SDS-polyacrylamide gel electrophoresis, the purified enzyme migrates as a single protein with apparent mass of 28 kDa. The molecular weight, as determined by electrospray mass spectral analysis, and the amino-terminal sequence are in accord with what was predicted from the DNA sequence. Steady state kinetic parameters, effects of pH, salts, and inhibition constants of several anti-folates have been determined.

The major surface antigen of Pneumocystis carinii, a life-threatening opportunistic pathogen in human immunodeficiency virus-infected patients, is an abundant glycoprotein that functions in host-organism interactions. A monoclonal antibody to this antigen is protective in animals, and thus this antigen is a good candidate for development as a vaccine to prevent or control P. carinii infection. We have cloned and sequenced seven related but unique genes encoding the major surface glycoprotein of rat P. carinii. Partial amino acid sequencing confirmed the identity of these genes. Based on Southern blot studies using chromosomal or restricted DNA, the major surface glycoproteins are the products of a multicopy family of genes. The predicted protein has an M(r) of approximately 123,000, is relatively rich in cysteine residues (5.5%) that are very strongly conserved, and contains a well conserved hydrophobic region at the carboxyl terminus. The presence of multiple related msg genes encoding the major surface glycoprotein of P. carinii suggests that antigenic variation is a possible mechanism for evading host defenses. Further characterization of this family of genes should allow the development of novel approaches to the control of this pathogen.

The opportunistic fungal pathogen Cryptococcus neoformans has two mating types, MATa and MAT alpha. The MAT alpha strains are more virulent. Mating of opposite mating type haploid yeast cells results in the production of a filamentous hyphal phase. The MAT alpha locus has been isolated in this study in order to identify the genetic differences between mating types and their contribution to virulence. A 138-bp fragment of MAT alpha-specific DNA which cosegregates with alpha-mating type was isolated by using a difference cloning method. Overlapping phage and cosmid clones spanning the entire MAT alpha locus were isolated by using this MAT alpha-specific fragment as a probe. Mapping of these clones physically defined the MAT alpha locus to a 35- to 45-kb region which is present only in MAT alpha strains. Transformation studies with fragments of the MAT alpha locus identified a 2.1-kb XbaI-HindIII fragment that directs starvation-induced filament formation in MATa cells but not in MAT alpha cells. This 2.1-kb fragment contains a gene, MF alpha, with a small open reading frame encoding a pheromone precursor similar to the lipoprotein mating factors found in Saccharomyces cerevisiae, Ustilago maydis, and Schizosaccharomyces pombe. The ability of the MATa cells to express, process, and secrete the MAT alpha pheromone in response to starvation suggests similar mechanisms for these processes in both cell types. These results also suggest that the production of pheromone is under a type of nutritional control shared by the two cell types.

Development of a transformation system for the fungal human pathogen Cryptococcus neoformans is an important prerequisite for the identification of genes involved in virulence. It has previously been reported that low-efficiency transformation can be achieved by using the cloned C. neoformans URA5 gene and ura5 mutants. The introduction of linearized URA5 vectors into C. neoformans resulted in unstable transformants which apparently harbored linear extrachromosomal DNA molecules. In this paper, the nature of these molecules is confirmed to be linear by exonuclease digestion. Recovery of the extrachromosomal DNA in Escherichia coli and sequence analysis demonstrates that repeats characteristic of telomeric DNA have been added to the ends of the introduced DNA. The recovered plasmids are capable of transforming at much higher efficiencies either in the supercoiled state (up to 200 transformants per microgram) or the linear state (up to 90,000 transformants per microgram).

Cryptococcus neoformans var. neoformans ura5 mutants were transformed with linearized or circular plasmids containing the C. neoformans orotidine monophosphate pyrophosphorylase gene. Following electroporation, randomly isolated transformants were analyzed for the mitotic and meiotic stability of uracil prototrophy. All stable transformants tested showed nonspecific ectopic integration. Uracil prototrophy in these transformants was stable through meiosis. Some of the stable transformants showed integration of both URA5 and vector sequences, while others lacked any vector sequences. Unstable transformants exhibited the presence of an autonomously replicating plasmid which had undergone significant sequence rearrangement. The autonomously replicating plasmid in the transformants was observed to be the same size or smaller than the transforming plasmid, was maintained in a linear form, and had acquired a genomic sequence(s) with homology to a sequence(s) on all the chromosomes. The conservation of a 300-bp sequence at the 5' end of the URA5 gene was observed in all the rearranged plasmids. These results suggest mechanisms of plasmid maintenance in C. neoformans that are different from those reported for other yeasts. The ura5 mutant was significantly less virulent than the wild type. The transformants did not recover virulence regardless of prototrophic stability.

A pair of congenic Cryptococcus neoformans var. neoformans strains, B-4476 (a mating type) and B-4500 (alpha mating type), that presumably differ only in mating type was constructed. This pair and their progeny, five alpha type and five a type, were tested for virulence in mice. In the parent strains as well as the progeny, alpha type was clearly more virulent than a type. In addition, death tended to occur earlier among the alpha-strain-infected mice that died than among the mice that died by infection caused by a strains. These data strongly suggest the genetic association of virulence with mating type in this human fungal pathogen.

Spontaneous mutants requiring uracil were isolated from both varieties of Cryptococcus neoformans by plating on 5-fluoroorotic acid (5-FOA) medium. Of the 36 strains tested (18 var. neoformans and 18 var. gattii), 24 (12 of each variety) generated 5-FOA-resistant cells requiring uracil for growth. Six of the 12 C. neoformans var. gattii strains produced ura3 cells while the remaining six strains produced ura5 cells. None of the 12 strains produced both ura3 cells and ura5 cells. All 12 isolates of var. neoformans, however, produced ura5 cells and one of them produced ura3 as well as ura5 cells. A genetic lesion in the URA5 gene of an isolate of C. neoformans var. gattii was confirmed by complement with the cognate URA5 gene of C. neoformans var. neoformans. The ura3 isolates were tentatively identified by their ability to grow on a medium containing uridine but not on a medium with orotic acid or orotidine. Enzymatic assays for orotidine-5'-phosphate decarboxylase activity confirmed the isolates to be ura3 mutants. Hybridization analysis of total DNA, digested with EcoRI or StuI and probed with pURA5g2, revealed the presence of only one copy of URA5 in the strains of either variety, regardless of the prevalence of ura5 mutants. Extensive polymorphism was observed in the restriction patterns of the fragments containing the URA5 locus. The prevalence of spontaneously arising ura3 mutants among the isolates of C. neoformans var. gattii, but not among the isolates of C. neoformans var. neoformans, is one more biological difference that distinguishes the two varieties.

Pneumocystis carinii dihydrofolate reductase (DHFR) expressed in Escherichia coli was purified to homogeneity in a single step using methotrexate-Sepharose affinity chromatography. The purified enzyme migrated as a single 24-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the first 26 amino acids from the N-terminus of the purified enzyme was in accord with that predicted from the DNA sequence. The enzyme showed a broad pH optimum with maximum activity over the pH range 6 to 7. The enzyme was activated by salts, with maximal twofold activation at 50 to 150 mM KCl and 50 to 200 mM NaCl. Urea at 2.5 M also increased the enzyme activity twofold. Kinetic analysis of the purified enzyme revealed that the Km values for dihydrofolate and NADPH were 1.8 and 1.4 microM, respectively, and that the kcat was 70 s-1. Inhibition studies verified that trimethoprim and pyrimethamine were poor inhibitors of P. carinii DHFR and showed little selectivity over the human DHFR. Trimetrexate and piritrexim were much more potent inhibitors of the P. carinii enzyme, but these inhibitors are also potent inhibitors of human DHFR.

A cDNA encoding Cryptococcus neoformans orotidine monophosphate pyrophosphorylase (OMPPase) has been isolated by complementation of the cognate Escherichia coli pyrE mutant. The cDNA was used as a probe to isolate a genomic DNA fragment encoding the OMPPase gene (URA5). By using electroporation for the introduction of plasmid DNA containing the URA5 gene, C. neoformans ura5 mutants could be transformed at low efficiency. Ura+ transformants obtained with supercoiled plasmids containing the URA5 gene showed marked mitotic instability and contained extrachromosomal URA5 sequences, suggesting limited ability to replicate within C. neoformans. Transformants obtained with linear DNA were of two classes: stable transformants with integrated URA5 sequences, and unstable transformants with extrachromosomal URA5 sequences.

Pulsed field gel electrophoresis was used to identify the chromosome-size DNA of Pneumocystis carinii, a major pathogen of immunocompromised patients. Thirteen chromosomes of rodent Pneumocystis carinii, ranging in size from 300 to 700 kilobases (kb), were identified. The minimum genome size for P. carinii, estimated on the basis of the sizes of chromosomes, is 7,000 kb. Genetic heterogeneity among different P. carinii isolates was documented by demonstration of chromosomal size variability. By hybridization studies, the genes for topoisomerase I, dihydrofolate reductase, rRNA, actin, and thymidylate synthase were mapped to single chromosomes of approximately 650, 590, 550, 460, and 350 kb, respectively. Hybridization studies further confirmed the genetic heterogeneity of P. carinii.

A cDNA for a potential tyrosine kinase-encoding mRNA was isolated from a mouse testis cDNA library. In a survey of eight mouse tissues, a transcript of 2.4 kilobases restricted to testis tissue was found. The mRNA encodes a 453-amino-acid protein of 51,383 daltons, the smallest tyrosine kinase protein ever described. RNA synthesized from the cDNA template directs the synthesis of a 51,000-Mr protein in a cell-free translation system. The carboxy-terminal 409 amino acids are 98 and 90% identical to the carboxy halves of the rat and human Fer proteins, respectively. This suggests that the cDNA represents an alternatively spliced testis-specific fer mRNA and is therefore termed by us ferT. On the basis of the appearance time of the fer mRNA in the testis of maturing neonatal mice, we speculate on the role played by this protein in the development of this organ.

Pneumocystis carinii is an intriguing organism found almost exclusively in the lungs. Very little is known about this organism's biologic or epidemiologic character. Over the past two decades, P. carinii has been recognized with increasing frequency as a cause of pneumonia in cancer patients, transplant recipients, and patients infected with the human immunodeficiency virus (HIV). With the increased number of cases of P. carinii pneumonia and a greater emphasis on studying this organism, sophisticated immunologic, metabolic, and molecular biologic tools have ben applied to enhance diagnosis, therapy, and prevention. Immunologic studies have identified specific antigens of human P. carinii, resulting in the development of new diagnostic tests and more specific serologic data. Metabolic studies have allowed screening and identification of new therapeutic and preventive drugs. The development of nucleic acid libraries has allowed enzymes and other proteins to be elaborated in large quantities, facilitating a wide range of studies. These new techniques have changed and will continue to change the ways that pneumocystis pneumonia is diagnosed, treated, prevented, and understood.

The thymidylate synthase (TS) gene from Pneumocystis carinii has been isolated from complementary and genomic DNA libraries and expressed in Escherichia coli. The coding sequence of TS is 891 nucleotides, encoding a 297-amino acid protein of Mr 34,269. The deduced amino acid sequence is similar to TS from other organisms and is most closely related to the enzyme from Saccharomyces cerevisiae with 65% identity. TS is found on a 330-kilobase-pair chromosome in P. carinii. While TS and dihydrofolate reductase reside on a single polypeptide chain in all protozoa studied to date, TS is not linked to dihydrofolate reductase in P. carinii. The TS gene shows the presence of four small intervening sequences, some of which interrupt the coding sequence in highly ordered structural regions of the protein. Heterologous expression of P. carinii TS in E. coli was accomplished by cloning the coding sequence into plasmid vectors under control of the lac and tac promoters. These constructs direct the synthesis of catalytically active enzyme to the extent of 2% of total soluble protein.

Pneumocystis carinii pneumonia is the most common opportunistic infection in AIDS, and accounts for significant morbidity and mortality in these and other immunocompromised patients. P. carinii is a eukaryotic microorganism of uncertain taxonomy that can infect numerous mammalian hosts. Developing from a small, unicellular 'trophozoite' into a 'cyst' containing eight 'sporozoites', its life cycle superficially resembles those seen both in the Protozoa and Fungi. Morphological and ultrastructural observations have lead some investigators to conclude that the organism is a protozoan, while others have felt that it more closely resembles a fungus. Phylogenetic relationships can be inferred from comparisons of macromolecular sequences. Small subunit ribosomal RNAs (16S-like rRNAs) are well-suited for this purpose because they have the same function in all organisms and contain sufficient information to estimate both close and distant evolutionary relationships. Phylogenetic frameworks based upon such comparisons reveal that the plant, animal and fungal lineages are distinct from the diverse spectrum of protozoan lineages. In this letter, phylogenetic analysis of Pneumocystis 16S-like rRNA demonstrates it to be a member of the Fungi.

The coding region for the Pneumocystis carinii small subunit rRNA (16S-like rRNA) contains 390 extra base pairs in a region that is highly conserved in other eukaryotic rRNA genes. The sequence has all the elements characteristic of group I self-splicing introns and interrupts the phylogenetically conserved helix proximal to the 3' terminus of the 16S-like rRNA. In vitro transcripts containing the putative group I intron were capable of undergoing a "self-splicing" reaction which required the presence of guanine nucleotide.

The primary structure of human insulin-like growth factor II receptor, predicted from the complementary DNA sequence, reveals a transmembrane receptor molecule with a large extracellular domain made up of fifteen repeat sequences and a small region homologous to the collagen-binding domain of fibronectin. The structural and biochemical features of the IGF-II receptor appear identical to those of the cation-independent mannose-6-phosphate receptor.

Pneumocystis carinii dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) cDNA sequences have been isolated by their ability to confer trimethoprim resistance to Escherichia coli. Consistent with the recent conclusion that P. carinii is a member of the Fungi, sequence analysis and chromosomal localization show that DHFR is neither physically nor genetically linked to thymidylate synthase. Expression of recombinant P. carinii DHFR in heterologous hosts provides an abundant source of the enzyme that may form a basis for the development of new therapies for this enigmatic pathogen. Studies with the recombinant enzyme show that trimethoprim is a very poor inhibitor of P. carinii DHFR and, in fact, is a more potent inhibitor of human DHFR.

Malignant lymphoma is a frequent complication of organ transplantation. It has been suggested that such tumors arise as a result of uncontrolled proliferation of Epstein-Barr virus-infected B lymphocytes in an immunosuppressed host. Although a few cases of posttransplant lymphomas in bone marrow transplantation have been shown to be of donor cell origin, no recipients of solid-organ transplants are known to have developed lymphomas arising from donor cells. In this report, a case of diffuse high-grade lymphoma that apparently arose in the allograft of a renal transplant recipient is described. DNA fingerprinting demonstrated the tumor to be of donor origin; Epstein-Barr sequences were absent. A therapeutic trial consisting of withdrawal of immunosuppressive agents and administration of acyclovir was unsuccessful. These data support the notion that donor cells can undergo malignant transformation in solid-organ transplant recipients, and such tumors need not carry EBV genetic material.

The formation of disulphide bonds is essential to the structure and function of proteins. These bonds rapidly form either cotranslationally or immediately post-translationally in the lumen of the endoplasmic reticulum. Native disulphide pairing for such proteins has been achieved in vitro; however, the rates of reassembly are slow and the conditions non-physiological. To account for these observations, Anfinsen et al. proposed that a 'disulphide interchange protein' was the in vivo catalyst of disulphide bond rearrangement. Other groups discovered an activity with similar characteristics that catalysed the reductive cleavage of insulin and may be associated with insulin degradation, although this result has been disputed. The enzyme involved, protein disulphide isomerase (PDI; EC 5.3.4.1), may be the in vivo catalyst of disulphide bond formation. Here we describe the sequence of cloned rat liver PDI complementary DNA which predicts a protein with two distinct regions homologous with Escherichia coli thioredoxin, a known cofactor in oxidation-reduction reactions. Each of these regions contains the presumed active site sequence Trp-Cys-Gly-His-Cys-Lys, suggesting that PDI, similar in action to thioredoxin, catalyses disulphide bond interchange via an internal disulphide-sulphydryl interchange. The cDNA predicts a signal peptide consistent with the view that PDI is a luminal endoplasmic reticulum protein. PDI messenger RNA, although ubiquitous, is more highly concentrated in secretory cells.

Hepatitis derived from hepatitis B virus (HBV) infection is endemic throughout the world, but it is particularly prevalent in Asia and Africa. In these areas, demographic studies show a strong coincidence between HBV infection (assayed by HBV antigenic markers) and the incidence of primary liver cancer. On these grounds, a causal link between HBV infection and primary hepatocellular cancer has been proposed. Recently, a human hepatoma cell line (PLC/PRF/5; Alexander cells) has been shown to produce hepatitis B surface antigen (HBsAg). We show here that the Alexander cell line contains at least six (four complete and two partial) hepatitis B viral genomes integrated into high molecular weight host DNA. An analysis using specific probes to fragments of the HBV genome suggests that integration of the virus in most cases occurs at the nicked cohesive end region of the virus. Expression of viral sequences using Northern blots demonstrates the presence of RNA transcripts specific for the surface antigen sequences of HBV DNA and the absence of detectable transcripts corresponding to the hepatitis B core antigen.

Stable transfectants of Chinese hamster ovary (CHO) cells were developed that expressed the protein encoded by a human insulin-like growth factor I (IGF-I) receptor cDNA. The transfected cells expressed approximately 25,000 high affinity receptors for IGF-I (apparent Kd of 1.5 X 10(-9) M), whereas the parental CHO cells expressed only 5,000 receptors per cell (apparent Kd of 1.3 X 10(-9) M). A monoclonal antibody specific for the human IGF-I receptor inhibited IGF-I binding to the expressed receptor and immunoprecipitated polypeptides of apparent Mr values approximately 135,000 and 95,000 from metabolically labeled lysates of the transfected cells but not control cells. The expressed receptor was also capable of binding IGF-II with high affinity (Kd approximately 3 nM) and weakly recognized insulin (with about 1% the potency of IGF-I). The human IGF-I receptor expressed in these cells was capable of IGF-I-stimulated autophosphorylation and phosphorylation of endogenous substrates in the intact cell. This receptor also mediated IGF-I-stimulated glucose uptake, glycogen synthesis, and DNA synthesis. The extent of these responses was comparable to the stimulation by insulin of the same biological responses in CHO cells expressing the human insulin receptor. These results indicate that the isolated cDNA encodes a functional IGF-I receptor and that there are no inherent differences in the abilities of the insulin and IGF-I receptors to mediate rapid and long term biological responses when expressed in the same cell type. The high affinity of this receptor for IGF-II also suggests that it may be important in mediating biological responses to IGF-II as well as IGF-I.