Combined pulmonary stenosis and insufficiency preserves myocardial contractility in the developing heart of growing swine at midterm follow-up.

This study was conducted to determine the effects of chronic combined pulmonary stenosis and pulmonary insufficiency (PSPI) on right (RV) and left ventricular (LV) function in young, growing swine. Six pigs with combined PSPI were studied, and data were compared with previously published data of animals with isolated pulmonary insufficiency and controls. Indexes of systolic function (stroke volume, ejection fraction, and cardiac functional reserve), myocardial contractility (slope of the end-systolic pressure-volume and change in pressure over time-end-diastolic volume relationship), and diastolic compliance were assessed within 2 days of intervention and 3 mo later. Magnetic resonance imaging was used to quantify pulmonary insufficiency and ventricular volumes. The conductance catheter was used to obtain indexes of the cardiac functional reserve, diastolic compliance, and myocardial contractility from pressure-volume relations acquired at rest and under dobutamine infusion. In the PSPI group, the pulmonary regurgitant fraction was 34.3 +/- 5.8%, the pressure gradient across the site of pulmonary stenosis was 20.9 +/- 20 mmHg, and the average RV peak systolic pressure was 70% systemic at 12 wk follow-up. Biventricular resting cardiac outputs and cardiac functional reserves were significantly limited (P < 0.05), LV diastolic compliance significantly decreased (P < 0.05), but RV myocardial contractility significantly enhanced (P < 0.05) compared with control animals at 3-mo follow-up. In the young, developing heart, chronic combined PSPI impairs biventricular systolic pump function and diastolic compliance but preserves RV myocardial contractility.     

Insecticidal proteins from Bacillus thuringiensis protect corn from corn rootworms.

Field tests of corn co-expressing two new delta-endotoxins from Bacillus thuringiensis (Bt) have demonstrated protection from root damage by western corn rootworm (Diabrotica virgifera virgifera LeConte). The level of protection exceeds that provided by chemical insecticides. In the bacterium, these proteins form crystals during the sporulation phase of the growth cycle, are encoded by a single operon, and have molecular masses of 14 kDa and 44 kDa. Corn rootworm larvae fed on corn roots expressing the proteins showed histopathological symptoms in the midgut epithelium.     

The evolution of titin and related giant muscle proteins

Titin and twitchin are giant proteins expressed in muscle. They are mainly composed of domains belonging to the fibronectin class III and immunoglobulin c2 families, repeated many times. In addition, both proteins have a protein kinase domain near the C-terminus. This paper explores the evolution of these and related muscle proteins in an attempt to determine the order of events that gave rise to the different repeat patterns and the order of appearance of the proteins. Despite their great similarity at the level of sequence organization, titin and twitchin diverged from each other at least as early as the divergence between vertebrates and nematodes. Most of the repeating units in titin and twitchin were estimated to derive from three original domains. Chicken smooth-muscle myosin light-chain kinase (smMLCK) also has a kinase domain, several immunoglobulin domains, and a fibronectin domain. From a comparison of the kinase domains, titin is predicted to have appeared first during the evolution of the family, followed by twitchin and with the vertebrate MLCKs last to appear. The so-called C-protein from chicken is also a member of this family but has no kinase domain. Its origin remains unclear but it most probably pre-dates the titin/twitchin duplication. Correspondence to: D.G. Higgins     

Detection of New Paternal Dystrophin Gene Mutations in Isolated Cases of Dystrophinopathy in Females

Duchenne muscular dystrophy is one of the most common lethal monogenic disorders and is caused by dystrophin deficiency. The disease is transmitted as an X-linked recessive trait; however, recent biochemical and clinical studies have shown that many girls and women with a primary myopathy have an underlying dystrophinopathy, despite a negative family history for Duchenne dystrophy. These isolated female dystrophinopathy patients carried ambiguous diagnoses with presumed autosomal recessive inheritance (limbgirdle muscular dystrophy) prior to biochemical detection of dystrophin abnormalities in their muscle biopsy. It has been assumed that these female dystrophinopathy patients are heterozygous carriers who show preferential inactivation of the X chromosome harboring the normal dystrophin gene, although this has been shown for only a few X:autosome translocations and for two cases of discordant monozygotic twin female carriers. Here we study X-inactivation patterns of 13 female dystrophinopathy patients—10 isolated cases and 3 cases with a positive family history for Duchenne dystrophy in males. We show that all cases have skewed X-inactivation patterns in peripheral blood DNA. Of the nine isolated cases informative in our assay, eight showed inheritance of the dystrophin gene mutation from the paternal germ line. Only a single case showed maternal inheritance. The 10-fold higher incidence of paternal transmission of dystrophin gene mutations in these cases is at 30-fold variance with Bayesian predictions and gene mutation rates. Thus, our results suggest some mechanistic interaction between new dystrophin gene mutations, paternal inheritance, and skewed X inactivation. Our results provide both empirical risk data and a molecular diagnostic test method, which permit genetic counseling and prenatal diagnosis of this new category of patients.     

Agrobacterium-Mediated Transformation of Subterranean Clover (Trifolium subterraneum L.)

We have developed a rapid and reproducible transformation system for subterranean clover (Trifolium subterraneum L.) using Agrobacterium tumefaciens-mediated gene delivery. Hypocotyl segments from seeds that had been allowed to imbibe were used as explants, and regeneration was achieved via organogenesis. Glucose and acetosyringone were required in the co-cultivation medium for efficient gene transfer. DNA constructs containing four genes encoding the enzymes phosphinothricin acetyl transferase, [beta]-glucuronidase (GUS), neomycin phosphotransferase, and an [alpha]-amylase inhibitor were used to transform subterranean clover. Transgenic shoots were selected on a medium containing 50 mg/L of phosphinothricin. Four commercial cultivars of subterranean clover (representing all three subspecies) have been successfully transformed. Southern analysis revealed the integration of T-DNA into the subterranean clover genome. The expression of the introduced genes has been confirmed by enzyme assays and northern blot analyses. Transformed plants grown in the glasshouse showed resistance to the herbicide Basta at applications equal to or higher than rates recommended for killing subterranean clover in field conditions. In plants grown from the selfed seeds of the primary transformants, the newly acquired gene encoding GUS segregated as a dominant Mendelian trait.     

'Muprints' of the lac operon demonstrate physiological control over the randomness of in vivo transposition

A method called Muprinting has been developed that uses PCR to generate a detailed picture of the bacteriophage Mu transposition sites in chosen domains of the bacterial chromosome. Muprinting experiments In Escherichia coli show that the frequency of phage integration changes dramatically near two repressor binding sites in the lac operon. When the lac operon was repressed, hotspots for Mu transposition were found near the O₁ and O₂ operators that are proposed to make a repression loop. When cells were grown in lactose, Mu transposition near these operators was greatly diminished. Striking changes In transposition frequencies were limited to the control region and were not found in a region of the lacZ gene lying beyond the O₂ operator. Muprints of the bgl operon showed a different pattern; hotspots for Mu transposition detected in sequences upstream of the bglC promoter when the operon was silenced changed when the operon became activated by mutation. By targeting transposition to the regulatory regions around non-expressed genes, Mu may demonstrate a self-restraint mechanism that allows the virus to move through its host genome without disrupting the functions that contribute to a healthy cell physiology.     

A protein complex mediating mRNA degradation in Escherichia coli

mRNA degradation in Escherichia coli is mediated by a combination of exo- and endoribolucleases. We present evidence for a multiprotein complex which includes at least two enzymes that play important roles in mRNA degradation: the exoribonuclease poly-nucleotide phosphorylase (PNPase) and the endorlbo-nuclease RNase E. An activity which impedes the processive activity of PNPase at stem-loop structures also appears to be associated with the complex. This complex is estimated to have a molecular mass of about 500 kDa and includes several additional poly-peptides whose functions are unknown. The identification of a complex which includes several activities associated with mRNA degradation has implications for the mechanisms and co-ordinated control of mRNA degradation.     

Analysis by fluorescence microscopy of the development of compartment-specific gene expression during sporulation of Bacillus subtilis.

The use of a fluorogenic substrate, 5-octanoylaminofluorescein-di-beta-D-galactopyranoside, for beta-galactosidase has made it possible to visualize enzyme activity in individual cells of sporulating populations of Bacillus subtilis by fluorescence microscopy. lacZ fusions to different sporulation-associated genes have been used to investigate the cell compartmentalization of gene expression during sporulation. A strain with a lacZ fusion to sspA, a gene which is transcribed by E-sigma G at a late stage of sporulation, displayed predominantly compartment-specific fluorescence. Expression of the early-expressed spoIIA locus, which includes the structural gene for sigma F, was seen not to be compartmentalized. Populations of strains with lacZ fusions to gpr and dacF, genes which are transcribed by E-sigma F at intermediate stages of sporulation, included some organisms showing uncompartmentalized fluorescence and others showing compartment-specific fluorescence; the proportion showing compartment-specific fluorescence increased in samples taken later in sporulation. Several possible explanations of the results obtained with gpr and dacF are considered. A plausible interpretation is that sigma F activity is initially not compartmentalized and becomes compartmentalized as sporulation progresses. The progression to compartmentalization does not require the activities of the sporulation-specific factor sigma E or sigma G but may require some product of sigma F activity.     

Effect of sunscreens,irradiance and resting periods on the germination ofMetarhizium flavoviride conidia

Chemical sunscreens were incorporated into oil formulations of conidia of two isolates of the entomopathogenic fungusMetarhizium spp. and the formulations exposed to simulated solar radiation. After 2 h exposure several sunscreens gave protection as demonstrated by conidial germination after 24 h incubation on gelatin plates at 26°C but only the formulations with Eusolex 8021 showed higher germination than the unprotected control after 48 h incubation. During 5h of exposure, Eusolex 8021 failed to offer significant protection as demonstrated by conidial germination after 48 h incubation. Conidial damage was proportional to the duration of radiation received. Allowing periods of darkness between exposures did not result in decreased loss of viability. Storing conidia, after exposure to simulated radiation, for 24 h prior to germination reduced their viability.     

Model for Cell Wall Growth of Streptococcus faecalis

In exponentially growing and dividing cells of Streptococcus faecalis, it is proposed that the leading edge of the annularly closing cross wall is the point of extension for both cross wall and peripheral wall. Peripheral wall extension is thought to be produced by the separation or splitting of the cross wall at its junction with peripheral wall. This results in the pushing of the equatorial wall bands, found on S. faecalis walls, to subsequatorial positions. These bands therefore mark the separation of old wall from new wall. Mesosomal formation was observed usually to precede cross wall initiation.