Effect of DNA concentration on transgenesis rates in mice and pigs.

A retrospective analysis of transgenesis rates obtained in seven pronuclear microinjection programs was undertaken to determine if a relationship existed between the amount of DNA injected and transgenesis rates in the pig. Logistic regression analysis showed that as the concentration of DNA injected increased from 1 to 10 ng/microl, the number of transgenics when expressed as a proportion of the number liveborn (integration rate) increased from 4% to an average of 26%. A similar relationship was found when the number of molecules of DNA injected per picolitre was analysed. No evidence was obtained to suggest either parameter influenced integration rate in mice when the same constructs were injected. The number of transgenics liveborn when expressed as a proportion of ova injected (efficiency rate), increased as DNA concentration increased up to 7.5 ng/microl and then decreased at 10 ng/microl for both species suggesting that at this concentration DNA (or possible contaminants) may have influenced embryo survival. The relationship between efficiency and the number of molecules injected per picolitre was complex suggesting that the concentration at which DNA was injected was a better determinant of integration and efficiency rates. In conclusion, the present study suggests that transgenes need to be injected at concentrations of between 5 and 10 ng/microl to maximise integration and efficiency rates in pigs.

Physeal stapling for idiopathic genu valgum.

Adolescent idiopathic genu valgum may cause anterior knee pain, patellofemoral instability, circumduction gait, and difficulty running. The purpose of this study was to evaluate and discuss what we consider to be an ideal treatment protocol using hemiphyseal stapling. We reviewed 76 patients (152 knees) who underwent hemiphyseal stapling for idiopathic adolescent genu valgum and were followed up to maturity. Clinical evaluation included assessment of gait, limb length, alignment, and patellofemoral stability. Radiographic evaluation included measurement of the distal femoral angle (DFA), the anatomic femoral tibial angle (FTA), and the mechanical axis (MA) before stapling, at the time of staple removal, and at skeletal maturity. After stapling, we noted improvement in gait, clinical symptoms, and all radiographic parameters. Our conclusion is that adolescent genu valgum may cause significant symptoms including anterior knee pain and gait problems. Hemiphyseal stapling addresses the anatomic malalignment, alleviating symptoms while offering a high degree of patient satisfaction. It is safe and effective, with no premature physeal closures noted in our series. The procedure, which is well tolerated, obviates the need for corrective femoral osteotomies.

The alpha-1,3-galactosyltransferase knockout mouse. Implications for xenotransplantation.

Organ xenografts in discordant combinations such as pig-to-man undergo hyperacute rejection due to the presence of naturally occurring human anti-pig xenoantibodies. The galactose alpha(1,3)-galactose epitope on glycolipids and glycoproteins is the major porcine xenoantigen recognized by these xenoantibodies. This epitope is formed by alpha(1,3)-galactosyltransferase, which is present in all mammals except man, apes, and Old World monkeys. We have generated mice lacking this major xenoantigen by inactivating the alpha(1,3)-galactosyltransferase gene. These mice are viable and have normal organs but develop cataracts. Substantially less xenoantibody from human serum binds to cells and tissues of these mice compared with normal mice. Similarly, there is less activation of human complement on cells from mice lacking the galactose alpha(1,3)-galactose epitope. These mice confirm the importance of the galactose alpha(1,3)-galactose epitope in human xenoreactivity and the logic of continuing efforts to generate pigs that lack this epitope as a source of donor organs.

A growth hormone agonist produced by targeted mutagenesis at binding site 1. Evidence that site 1 regulates bioactivity.

Growth hormone (GH) is believed to signal by dimerizing its receptor through two binding sites on the hormone. Previous attempts to increase the biopotency of GH by increasing its site 1 affinity have been unsuccessful, which has led to a bias toward engineering site 2 interactions in the quest for creation of super agonists. Here we report that increasing site 1 affinity can markedly increase proliferative bioactivity in FDC-P1 cells expressing full-length GHR. In contrast, we find three site 1 mutants with affinities for site one similar to or greater than wild type GH, which have markedly decreased bioactivity. Through crystal structure analysis of the receptor interactive regions of these GH analogues, we are able to suggest why previous mutagenesis on human GH failed to improve biopotency, and thus provide a new avenue for GH and cytokine agonist design.

Evidence for involvement of the carboxy terminus of helix 1 of growth hormone in receptor binding: use of charge reversal mutagenesis to account for calcium dependence of binding and for design of higher affinity analogues.

In this study we have demonstrated that the C-terminus of helix 1 of porcine GH (pGH) is a receptor-interactive region, thus extending the current binding site model of GH. This was achieved by introducing charge reversal mutations into this region of pGH, which influenced receptor affinity and Ca2+ dependence of binding. The first mutant (R34E pGH, conversion of Arg 34 to Glu) introduced a putative Ca2+ binding site which is present in human GH (hGH) [Barnard et al. (1989) J. Theor. Biol. 140, 355-367] and sits opposite E220 of receptor subunit 1. This mutant exhibited increased Ca2+ dependence of receptor binding but even at optimal Ca2+ did not display higher than wild-type affinity. Introduction of a second Ca2+ binding site adjacent to the first by a second charge reversal (K30E R34E pGH) further increased Ca2+ dependence of binding and also increased affinity for the rabbit GH receptor (2.4 +/- 0.4)-fold relative to wild-type pGH at optimal Ca2+. Equilibrium dialysis and Scatchard analysis of binding of 45Ca2+ to pGH and K30E R34E pGH revealed two Ca2+ binding sites on wild-type pGH and an additional two Ca2+ binding sites on the K30E R34E pGH mutant (Kd 0.5-0.8 mM), as predicted. A third partial charge reversal mutant in the fourth helix (H170D) also led to enhanced Ca2+ dependence of binding, supporting our proposal that E34 and D170 are responsible for the Ca2+ dependence of hGH binding to the rabbit GH receptor. Examination of the crystal structure shows that E34 and D170 are in close proximity and would interact repulsively with a cluster of acidic residues on the receptor consisting of E126, E127, and E220 unless neutralized by Ca2+ or an introduced basic residue. Accordingly, charge reversal at the adjacent pGH residue E33 (E33K pGH) led to a Ca2+ independent (3.0 +/- 0.4)-fold increase in affinity of binding. As well as extending the binding site model of GH, these studies provide a mechanistic explanation for the unique Ca2+ dependence of hGH binding to the rabbit GH receptor. They also indicate that charge reversal can be used to design higher affinity GH analogues and could assist in the mapping of interactive regions in ligand-receptor complexes generally.

Postoperative return of motion in anterior cruciate ligament and medial collateral ligament injuries. The effect of medial collateral ligament rupture location.

Twenty consecutive patients with combined anterior cruciate/medial collateral ligament injuries were analyzed to determine if a correlation exists between the location of medial collateral ligament disruption and postoperative return of motion. All patients were treated operatively by autogenous patellar tendon anterior cruciate ligament reconstruction and primary medial collateral ligament repair. The mean followup was 379 days. The patients (12 men and 8 women; mean age, 23 years) were divided into two groups based on the location of superficial medial collateral ligament rupture. Group P consisted of 13 patients with lesions at or proximal to the joint line; Group D consisted of 7 patients with disruptions distal to the joint line. Group D patients had a more rapid return of motion for both flexion and extension. At the conclusion of followup, patients from Group D also achieved 8 degrees more flexion and 3 degrees more extension. There were eight additional procedures performed on five patients, all from Group P, required to treat difficulty regaining motion. Among these patients with anterior cruciate/medial collateral ligament injuries there are two distinct groups, each with different prognoses related to return of motion based on the location of the medial collateral ligament disruption. We suggest that patients with double-ligament injuries, where the medial collateral ligament lesion is proximal, should be managed very aggressively to regain motion.

Identification of molecules involved in the 'early pregnancy factor' phenomenon.

An isolated preparation from ovine placental extracts which was active in the rosette inhibition assay mimicking the activity of the so-called 'early pregnancy factor' (EPF) has been shown to contain a 12 kDa polypeptide which could be partially resolved from low-molecular-weight active moieties. N-terminal amino acid sequence analysis of the polypeptide indicated that it was ovine thioredoxin, an identification confirmed by isolation and complete sequence analysis of the corresponding cDNA. The cDNA for human thioredoxin was expressed in Escherichia coli and the recombinant protein isolated and purified. Pure recombinant thioredoxin alone did not induce the expression of increased rosette inhibition titres (RITs) when tested in the rosette inhibition assay; but, when tested in combination with cell stimuli such as platelet-activating factor (PAF) or serum, it allowed the expression of increased RITs where none was achieved in its absence. Thioredoxin acted in the assay to reverse a refractory state normally induced by these stimuli, allowing lipoxygenase-dependent moieties also induced by the stimuli to exert their effects, resulting in the expression of increased RITs. Antibodies to recombinant thioredoxin removed from pregnancy sera the capacity to induce increased RITs, i.e. to express EPF activity, thus establishing a role for thioredoxin or thioredoxin-like proteins and associated molecules in the mechanisms which allow pregnancy sera to induce increased RITs. Based on a consideration of these and other results, a new model for the study of the EPF phenomenon is presented and discussed.

Transcription from the intron-containing chicken histone H2A.F gene is not S-phase regulated.

The nucleotide sequence of an 8.2 kb BamHI fragment containing the entire chicken histone H2AF gene has been determined. Unlike the majority of histone genes, the coding region is interrupted by four intervening sequences. While sequencing the 8.2 kb BamHI fragment it was found that the promoter and first exon of an unidentified non-histone gene lies immediately downstream of the H2AF gene. Studies of H2AF gene transcription show that, unlike the major core and H1 histone genes, it is not coupled to DNA synthesis.

Lifetime and six-month prevalence of psychiatric disorders among sentenced female offenders.

The authors determined the six-month and lifetime prevalence of psychiatric disorders among 100 consecutively admitted female offenders to a prison, using Diagnostic Interview Schedule (DIS Version III) and found high prevalence rates of schizophrenia, major depression, substance use disorders, psychosexual dysfunction, and antisocial personality disorders. The prevalence rates of these disorders were significantly higher than those of the general population. The authors note the implications of their findings for treatment of women within the correctional system.

Characterization of the chicken histone H1 gene complement. Generation of a complete set of vertebrate H1 protein sequences.

Sequence analysis of four chicken H1 histone genes described here completes the characterization of the full complement of six H1 genes in the chicken genome. Each of the six genes codes for a different H1 protein sequence, and these range in size from 217 to 224 amino acids. The proteins are distinct in sequence from the H1-related chicken H5 protein and appear to be analogous to the standard somatic mammalian H1 subtypes. The protein sequence data deduced from the genes represent the first complete set of vertebrate H1 protein sequences. Comparison of the chicken H1 gene noncoding sequences with each other and with H1 gene sequences from other organisms reveals conservation of an H1 gene-specific element, a G-rich element, and histone gene-specific 3' elements. Additional sequences are conserved between H1 genes of the chicken and other vertebrates. Comparisons also reveal variation in promoter and 3' elements between chicken genes that could play a role in the differential expression of H1 gene protein products.

Depression--medical utilization and somatization.

We screened 147 primary care patients for depression using depression rating scales and a psychiatric interview. In the one year after screening, the patients with depression visited and phoned their physicians more frequently and had more medical evaluations than the nondepressed control group. The patients with depression were more likely to have nonspecific or vague complaints and psychophysiologic or depressive symptoms than the control group; their family physicians during this same period were more likely to diagnose a psychophysiologic problem.

A unique element resembling a processed pseudogene.

We describe a unique DNA element with structural features of a processed pseudogene but with important differences. It is located within an 8.4-kilobase pair region of chicken DNA containing five histone genes, but it is not related to these genes. The presence of terminal repeats, an open reading frame (and stop codon), polyadenylation/processing signal, and a poly(A) rich region about 20 bases 3' to this, together with a lack of 5' promoter motifs all suggest a processed pseudogene. However, no parent gene can be detected in the genome by Southern blotting experiments and, in addition, codon boundary values and mid-base correlations are not consistent with a protein coding region of a eukaryotic gene. The element was detected in DNA from different chickens and in peafowl, but not in quail, pheasant, or turkey.

Inverted duplication of histone genes in chicken and disposition of regulatory sequences.

Sequence analysis of an 8.4 kb fragment containing five chicken histone genes shows that an H4-H2A gene pair is duplicated and inverted around a central H3 gene. A left and right region, each of 2.1 kb are 97% homologous and the boundaries of homology coincide with ten base pair repeats. These boundary regions also contain highly conserved gene promoter elements, suggesting that interaction of transcriptional machinery with histone genes may be connected with recombination in promoter regions, resulting in the inverted duplication structure seen in this cluster.

Ribosomal gene reiteration in a marsupial species with an X-linked nucleolar organizer.

The number of ribosomal cistrons in somatic cells of males and females of Macropus eugenii have been estimated using RNA/DNA hybridization in 70% (v/v) formamide. The male has one X-linked active nucleolar organizer and the female two and while the number of ribosomal cistrons is variable between the four males examined (31-65 copies), it is substantially less than the number present in the three females (94-103 copies). There is, therefore, no evidence of dosage compensation by amplification.

Depression in a sample of 9-year-old children, Prevalence and associated characteristics.

We investigated the prevalence of depression in a sample of 9-year-old children from the general population being studied longitudinally. Current point prevalences of major and minor depressive disorder were estimated at 1.8% and 2.5%, respectively. A comparison of children with depression and a nondepressed group disclosed no significant differences by sex, nor any significant association between depression and socioeconomic status, teacher reports of behavior problems, and cognitive or motor development. The children with current depression were reported by a parent to have had a history of more behavioral problems, had been referred more often for assessment or treatment of behavioral or emotional problems, and had more negative self-perceptions of their academic ability. The results suggested that parents may be more sensitive than teachers to the behavior problems exhibited by depressed children.

The chicken H5 gene is unlinked to core and H1 histone genes.

An H5 cDNA clone was used to select H5 genomal recombinants from a chicken Charon 4A library. DNA sequence analysis shows that the H5 gene contains no introns. Putative 5' promoter elements and a 3' polyadenylation site are present within the 1.8 kb of DNA examined. Analysis of 41 kb of DNA surrounding the H5 gene shows that it is not closely linked to either H1 or core histone genes.Images

Independently evolving chicken histone H2B genes: identification of a ubiquitous H2B-specific 5' element.

The DNA sequence of two chicken histone H2B genes has been determined. Both genes code for the same H2B subtype. Except for conserved "promoter" elements, the sequences 5' to the protein coding regions are completely divergent, indicating that the genes are distantly related and are not evolving in concert. This presents an ideal situation for sequence comparisons. We have discovered a 13 bp, H2B specific homology block, 5' CTCATTTGCATAC 3' located close to the "TATA box". This motif is conserved in all H2B gene leader regions so far sequenced. One of the H2B genes is closely linked, in a divergent arrangement, to an H2A gene, and sequence data suggests that the linked genes share promoter elements.

Chicken histone H5 mRNA: the polyadenylated RNA lacks the conserved histone 3' terminator sequence.

Using 3 overlapping cDNA clones we have determined the nucleotide sequence of chicken histone H5 mRNA. The mRNA does not contain the 23 base conserved sequence element that is present at the 3' end of cell-cycle regulated histone mRNAs. Although the RNA is polyadenylated it lacks the 3' AAUAAA sequence.