Unsuccessful treatment of chronic norovirus infection with enteral immunoglobulin in patients with common variable immunodeficiency, case report.

Background: Norovirus gastroenteritis is commonly an acute infection that lasts 2-3 days, but in immunocompromised patients norovirus can cause a chronic gastroenteritis lasting for years. Norovirus replicates in the gastrointestinal tract, but the pathway of viral clearance is not yet known. Promising results of enterally administered immunoglobulin in the treatment of chronic norovirus gastroenteritis in immunocompromised patients have previously been published. Case presentation: We report two individuals with common variable immunodeficiency and chronic debilitating norovirus gastroenteritis. Both patients were treated with enterally administered immunoglobulin via a duodenal feeding tube as other treatment modalities have been unsuccessful. The patients did not experience any immediate or long-term benefit of enterally administered immunoglobulin. Conclusion: Despite previous case reports of successful treatment of chronic norovirus infection among immunocompromised patients with enterally administered immunoglobulin, these two patients experienced no benefit of the treatment. This demonstrates the need for further research in treatment of chronic norovirus infection in immunocompromised patients.

Nucleotide sequence of bacteriophage T4 gene 23 and the amino acid sequence of its product.

We have determined the nucleotide sequence of gene 23 of bacteriophage T4 by the methods of Maxam and Gilbert and of Sanger. The identities of approximately 80% of the amino acid residues of the major capsid protein which is encoded by gene 23 were determined additionally by Edman degradation of the intact protein and its peptides. Fifteen gene 23 amber mutation sites have been located within the sequence, and the 3' transcription termination site for genes 21, 22 and 23 has been identified.

Cats as an aid to teaching genetics.

I have used an exercise involving domestic cats in the General Genetics course at the University of Nebraska-Lincoln for the past 5 years. Using a coherent set of traits in an organism familiar to the students makes it easy to illustrate principles of transmission and population genetics. The one-semester course consists primarily of sophomores and juniors who have either taken a one-semester introductory biology course, a one-semester cell biology course, or have a strong high school biology background. The students are given a handout and asked to determine the genotype at seven unlinked loci of at least one cat. To fill out the form, the students have to grasp such concepts as dominance, incomplete dominance, temperature-sensitive mutations, epistatic interactions, sex linkage, and variable expressivity. Completing the form reinforces these concepts as they observe the cat's phenotype and fill in the genotype. I then analyze the collected data and use it in my lectures on population genetics to illustrate the Hardy-Weinberg equilibrium, calculate allele frequencies, and use statistics. This allows the students to look at population genetics in a very positive light and provides concrete examples of some often misunderstood principles.

A recombinational hotspot at the triplo-lethal locus of Drosophila melanogaster.

In the genome of Drosophila melanogaster there is only one locus, Tpl, that is triplo-lethal; it is also haplo-lethal. Previous work has identified 3 hypomorphic alleles of Tpl which rescue animals carrying a duplication of Tpl, but which are not dominant lethals as null mutations or deficiencies would be. We have found that all three hypomorphic alleles act as site-specific hotspots for recombination when heterozygous with a wild-type homolog. Recombination between the flanking markers ri and Ki is increased 6.5-10.5-fold in the presence of Tpl hypomorphic alleles. The increased recombination was found to occur between Tpl and Ki, while recombination in other adjacent regions is unchanged. The use of isogenic Tpl+ controls, and the use of flanking intervals in the mutant chromosomes allows us to rule out the interchromosomal effect as a cause. We have also observed premeiotic recombination occurring at the Tpl hypomorphic alleles in male heterozygotes. We hypothesize that transposons are responsible for both the hypomorphic phenotype and the high frequency of recombination.

The unusual spectrum of mutations induced by hybrid dysgenesis at the Triplo-lethal locus of Drosophila melanogaster.

The Triplo-lethal locus (Tpl) is unique in its dosage sensitivity; no other locus in Drosophila has been identified that is lethal when present in three doses. Tpl is also haplo-lethal, and its function is still a mystery. Previous workers have found it nearly impossible to mutationally inactive Tpl other than by completely deleting the chromosomal region in which Tpl resides (83DE). We have utilized P-M hybrid dysgenesis in an effort to obtain new mutations of Tpl. We recovered 19 new duplications of Tpl, 15 hypomorphic mutations of Tpl (a previously rare class of mutation), and no null mutations. Surprisingly, 14 of the 15 hypomorphic alleles have no detectable P element sequences at the locus. The difficulty in recovering null mutations in Tpl suggests that it may be a complex locus, perhaps consisting of several genes with redundant functions. The relative ease with which we recovered hypomorphic alleles is in sharp contrast to previous attempts by others to mutagenize Tpl. A higher mutation rate with hybrid dysgenesis than with radiation or chemicals also suggests a peculiar genetic organization for the locus.

The Triplo-lethal locus of Drosophila: reexamination of mutants and discovery of a second-site suppressor.

In the genome of Drosophila melanogaster there is a single locus, Triplo-lethal (Tpl), that causes lethality when present in either one or three copies in an otherwise diploid animal. Previous attempts to mutagenize Tpl produced alleles that were viable over a chromosome bearing a duplication of Tpl, but were not lethal in combination with a wild-type chromosome, as deficiencies for Tpl are. These mutations were interpreted as hypomorphic alleles of Tpl. In this work, we show that these alleles are not mutations at Tpl; rather, they are dominant mutations in a tightly linked, but cytologically distant, locus that we have named Suppressor-of-Tpl (Sul(Tpl)). Su(Tpl) mutations suppress the lethality associated with three copies of the Triplo-lethal locus and are recessive lethal. We have mapped Su(Tpl) to the approximate map position 3-46.5, within the cytological region 76B-76D.

Suppression of a lethal trisomic phenotype in Drosophila melanogaster by increased dosage of an unlinked locus.

One of the most extreme examples of gene dosage sensitivity is the Triplo-lethal locus (Tpl) on the third chromosome of Drosophila melanogaster, which is lethal when present in either one or three copies. Increased dosage of an unlinked locus, Isis, suppresses the triplo-lethal phenotype of Tpl, but not the haplo-lethal phenotype. We have mapped Isis to the X chromosome region 7E3-8A5, and shown that the suppression is a gene dosage effect. Altered dosage of Isis in the presence of two copies of Tpl has no obvious effects. By examining the interactions between Isis dosage and Tpl we suggest that Isis does not directly repress Tpl expression, but acts downstream on the triplo-lethal phenotype of Tpl.

A peritrophin-like protein expressed in the embryonic tracheae of Drosophila melanogaster.

We have cloned and sequenced a cDNA from Drosophila melanogaster that encodes a protein homologous to the peritrophins, a family of chitin-binding proteins from the peritrophic matrix of insects. Unexpectedly, the gene, Gasp, is expressed in the embryonic tracheae. We suggest that this family of proteins may be present in other tissues than the peritrophic matrix, particularly where nutrient or gas exchange are important, and/or where invasion by parasites or viruses is possible. We have also mapped two similar genes that had been sequenced by the Berkeley Drosophila Genome Project, and find that these three very similar genes are not clustered, but are located on three different chromosomes.

Bacteriophage lambda-based expression vectors.

Bacteriophage lambda has been in use as a cloning vector for over 25 years, and has been used extensively as an expression vector. The efficiency of packaging and infection, and the simplicity of plaque screening are advantages of lambda as a cloning vector. A number of ingenious modifications help overcome the disadvantages associated with its mode of growth and its size. Some lambda vectors have been designed to be readily converted into plasmids or phagemids, and there are a variety of promoters and fusions that can be used to drive expression of foreign genes. Screening lambda libraries with antibodies or ligands is a powerful way of identifying novel genes.

Reappraisal of the human ocular growth curve in fetal life, infancy, and early childhood.

AIMS: The aim of this study was to find an algorithm of better fit for early eye growth than the linear regression usually advanced. METHODS: The analysis is based on previously published around term data, the main material being axial ultrasound measurements in preterm (n = 101) and full term infants (n = 25). The postconceptional age of the infants ranged between 36 and 54 weeks. Previously published Danish data from eyes of aborted fetuses were also used, as were averaged values from the literature regarding eye size at age 1 year (20 mm), 3 years (22 mm), and a presumed 13 year endpoint of 23 mm. RESULTS: A second order exponential function fitted with the basic data within a standard deviation of 2%. CONCLUSIONS: A simple symbolic expression and tabulated values for eye growth in infancy and childhood were derived. This is clearly of practical value, for example, when following the development of eyes treated for congenital glaucoma or assessing other developmental anomalies and early eye diseases.

Factors affecting efficacy of methionine hydroxy analogue for chicks fed practical diets.

The relative values of the calcium salt of the hydroxy analogue of methionine (MHA) and L-methionine were studied in chick feeding tests with five practical type diets deficient in the sulfur amino acids. Performance was improved with the addition of either substance to each of the five diets. Methionine was slightly more effective at low levels of addition to the four diets primarily deficient in methionine. The two supplements were equally effective in one diet based on corn, soybean, and meat and bone meals, a diet with a wider methionine/cystine ratio. Both the practical and highly purified amino acid diets thus showed similar differences in efficacy of the two products as the methionine/cystine ratio of the diet changed. Methionine also was more effective than MHA in overcoming the deleterious effects produced by the addition of ethionine to a practical type diet.

Factors affecting efficacy of methionine hydroxy analogue for chicks fed amino acid diets.

Chicks were fed mixtures of methionine, cystine, and the calcium salt of the hydroxy analogue of methionine (MHA) in a diet based on a mixture of amino acids. Rate of gain with the basic amino acid diet containing a mixture of methionine and cystine was about 90% of the rate noted with a practical type diet. Efficacy of MHA depended on its level in the diet and on the levels of methionine and cystine fed with it. It was least effective when fed as the only sulfur amino acid or when fed with cystine. When fed with methionine it had intermediate value and was most efficacious when fed with a mixture of methionine and cystine. Essentially it was fully effective when it provided 25% of the sulfur amino acids with the remainder as equal parts of methionine and cystine. Replacing part of the cystine in a mixture of cystine and MHA with methionine resulted in a marked improvement in performance with L-methionine being slightly more effective than D-methionine.

Progesterone exposure of seasonally anoestrous ewes alters the expression of angiogenic growth factors in preovulatory follicles.

Small-dose, multiple injections of GnRH given to seasonally anoestrous ewes induce final stages of the preovulatory follicle development, but result in an high incidence of defective CL unless animals are primed with progesterone, which completely eliminates luteal dysfunction. Progesterone priming upregulates luteal vascularization; however, its effect on follicular angiogenesis is poorly understood. This study tested the hypothesis that progesterone priming of seasonally anoestrous ewes treated with dose multiple injections of GnRH eliminates defective luteal function by altering the expression of vascular endothelial growth factor (VEGF), VEGF receptor-2, angiopoietin (ANG)-1, ANG-2, and TIE-2 during early and late preovulatory follicle development. Ten seasonally anoestrous ewes were given 20 mg of progesterone im 3 days before the start of GnRH treatment; 10 other animals served as controls. Intravenous injections of 500 ng GnRH were given to all animals every 2 hours for 28 hours, followed at 30 hours with a 300-µg GnRH bolus injection to synchronize the preovulatory LH surge. Ovaries were collected at 24 and 46 hours after the start of GnRH treatment. Small (2-2.5 mm) and large (>2.5 mm) follicles were analyzed for protein and mRNA expression of the angiogenic factors using immunohistochemistry and in situ hybridization assays. Progesterone priming did not have an influence on angiogenic factor levels in small follicles. However, progesterone-primed animals showed significantly (P ≤ 0.05) higher levels of VEGF, VEGFR-2, ANG-1, and ANG-2 in large follicles compared with nonprimed ones. These data suggest that progesterone priming alters the expression of angiogenic factors in large preovulatory follicles, ensuring adequate luteal development and function.

Progesterone exposure of the preovulatory follicle in the seasonally anestrous ewe alters the expression of angiogenic growth factors in the early corpus luteum.

Gonadotrophin releasing hormone (GnRH)-induced ovulation in seasonally anestrous ewes is associated with a high incidence of defective corpora lutea (CL), which can be completely eliminated by priming ewes with progesterone before GnRH treatment, but the physiological basis of this has remained elusive. This study tested the hypothesis that progesterone priming eliminates defective luteal function by altering the expression of Vascular Endothelial Growth Factor (VEGF), its receptor VEGFR-2, and angiopoietin (ANG)-1, ANG-2 and their receptor TIE-2 in the early CL. Fifteen seasonally anestrous ewes were treated by i.m. injection with 20 mg of progesterone 3 days before the start of GnRH treatment, while another 15 animals served as controls. Intravenous injections of 500 ng GnRH were given to all the ewes every 2 h for 28 h, followed by a 300 µg GnRH bolus injection to synchronize the preovulatory luteinizing hormone (LH) surge. Corpora lutea were collected 1, 2 and 4 days after ovulation and analyzed for protein and mRNA expression of VEGF, VEGFR-2, ANG-1, ANG-2 and Tie-2 using Western Immunoblotting and in situ hybridization. VEGF, VEGFR-2 and ANG-1 expression was significantly higher (P ≤ 0.05) in the CL of progesterone-primed animals compared to non-primed ones. However, no differences were observed in the ANG-2 or Tie-2 expression levels between the two treatment groups. These data suggest that progesterone priming of the preovulatory follicle alters the expression of some angiogenic growth factors in the early CL, leading to greater vascular stability and thereby normal luteal function.

T4 late transcripts are initiated near a conserved DNA sequence.

Bacteriophage T4 late transcription is unusual, among prokaryotes, in its complexity. Late transcription requires the host RNA polymerase, the products of T4 genes, 33, 45 and 55, and other small polypeptides, the genes of which have not been identified. In addition the DNA template must be "competent' for late transcription. First the DNA must contain the substituted base 5-hydroxymethyl cytosine in place of cytosine (this requirement is eliminated by a mutation in the T4 alc gene). Second, the DNA must be replicating, although late transcription can be uncoupled from DNA replication by mutations in the T4 genes coding for DNA ligase (gene 30) and DNA exonuclease (gene 46). We report here the location of the initiation sites of the messenger RNAs (mRNAs) synthesized in vivo from four late genes (genes 21, 22, 23 and 36) by S1 nuclease mapping and we have determined the DNA sequences at these sites. We have found strong homology to the sequence TATAAATACTATT immediately upstream from the 5' ends of the late messages and we suggest that this sequence is specifically recognized by the complex responsible for late transcription. Also, we have examine gene 23 mRNA synthetized in the absence of DNA replication using the 30- 46- mutant described above and find that it is identical to the true late transcript synthesized in normal infections.

Ribosomal protein S14 is not responsible for the Minute phenotype associated with the M(1)7C locus in Drosophila melanogaster.

A locus associated with a severe Minute effect has been mapped at 7C on the X chromosome of Drosophila melanogaster. Previous work has suggested that this Minute encodes ribosomal proteins S14A and S14B. We have made a chromosomal deficiency that removes the S14 ribosomal protein genes, yet does not display the Minute phenotype. These data suggest that the S14 genes do not actually correspond to the Minute locus.

A novel RNA helicase gene tightly linked to the Triplo-lethal locus of Drosophila.

The Triplo-lethal (Tpl) locus of Drosophila is the only known locus which is lethal when present in three copies rather than the normal two. After recovering a hybrid-dysgenesis-induced mutation of Tpl we used a rapid combination of transposon tagging, chromosome microdissection and PCR to clone the P element that had transposed into the Tpl region. That P element is located within the gene for a new and unique member of the RNA helicase family. This new helicase differs from all others known by having glycine-rich repeats at both the amino and carboxyl termini. Curiously, genetic analysis shows that the P element inserted into this gene is not responsible for the Tpl mutant phenotype. We present possible explanations for these findings.

Effect of transferrin saturation on iron delivery in rats.

Studies were carried out in rats to evaluate the release of iron from monoferric and diferric transferrin. Isoelectric focusing was carried out on plasma tagged at low and high saturations, and it was shown that these represented primarily monoferric and diferric transferrin, respectively. In vivo studies with these tagged plasmas were carried out on normal, iron-deficient, and hypertransfused rats. In all three groups there was a greater in vivo uptake of iron from diferric transferrin by all tissues monitored. Though the amount varied in different animals, the ratio of uptake between diferric and monoferric transferrin iron varied between 1.56 and 2.10 in the erythron and between 2.38 and 2.65 in the liver. These studies indicate that changes in transferrin saturation, by changing the proportion of monoferric to diferric transferrin iron, changed the amount of iron released to tissues.