DNA probe analysis for carrier detection and prenatal diagnosis of Duchenne muscular dystrophy: a standard diagnostic procedure.

Thirteen marker loci localised on the short arm of the X chromosome are available for use in genetic studies for Duchenne muscular dystrophy (DMD). This large number of probes detecting about 20 RFLPs encouraged us to set up a standard procedure using a sequence of selected probes and restriction enzymes for the diagnosis of DMD families. The application of DNA probe analysis for carrier detection and prenatal diagnosis, involving 61 pedigrees of both familial and isolated cases, has yielded the following results. Carrier detection using flanking markers was possible in more than 75% of the cases (104 out of 136 females) with a reliability of better than 98%. Prenatal diagnosis was possible in 95% of the cases (65 out of 68 proven carriers or women at risk). Twenty-three prenatal diagnoses were performed on male fetuses; 13 appeared to have a low risk for DMD (less than 1%) and thus the pregnancies continued. Seven have since come to term and the male infants have normal CK levels. The genetic distances of the loci relative to the DMD locus and their order on the short arm of the X chromosome were deduced from our total DMD family material and are not significantly different from those reported earlier. For 754 (DXS84) we found a genetic distance of 5 cM with a lod score of +12.4 and 95% confidence limits between 2 and 12 cM. Similar data were obtained for pERT87 (DXS164), suggesting that in our family material both loci are tightly linked. Multiply informative recombination showed that both 754 and pERT87 map proximal to the DMD mutations in the cases studied. The high frequency of DMD mutations and its relation to the observed instability in this part of the genome will be discussed. Unequal crossing over is proposed as one of the mechanisms contributing to the high mutation frequency.     

Is semiquantitative culture of central vein catheter tips useful in the diagnosis of catheter-associated bacteremia?

Semiquantitative culturing of catheter tips has been used as an index of catheter-related bacteremia. As the sensitivity and predictive values of this test have not been determined, we studied 780 tips from central vein catheters inserted into 440 critically ill patients in an intensive care unit. The results were correlated with clinical data for 30 bacteremic episodes which occurred in these patients, 14 of which were catheter related. When five or more colonies per plate were taken as a positive result, the sensitivity of the method was 92%, and the specificity was 83%. Although the predictive value of a negative result was excellent (99.8%), the predictive value of a positive result was low (8.8%) in our patient population, which had a relatively low incidence of catheter-related bacteremia (2%). We conclude that a semiquantitative culture technique is useful in the diagnosis of bacteremia associated with central vein catheters.     

The use of linked DNA polymorphisms for genotype prediction in families with Duchenne muscular dystrophy.

Two DNA restriction fragment length polymorphisms show genetic linkage to the Duchenne muscular dystrophy locus on the short arm of the X chromosome. Examples are given of families in which these polymorphisms can be used in the prediction of genotype for this disorder.     

Human herpes virus 6 and endogenous biotin in salivary glands.

AIMS: To detect the presence of human herpes virus 6 (HHV6) and endogenous biotin in paraffin wax embedded and frozen salivary glands. METHODS: Two stage indirect and streptavidin-biotin immunoperoxidase techniques were used to visualise the antigens. RESULTS: HHV6 could not be shown in any of the tissues. However, considerable endogenous biotin antigenicity was detected in the glandular elements of the paraffin wax embedded material. CONCLUSIONS: Results obtained with avidin-biotin detection systems should be interpreted with caution, especially when glandular epithelium is being stained. This may apply to both immunoperoxidase and in situ hybridisation techniques. The use of an anti-biotin antibody as a standard control should be considered.     

Pseudomonas aeruginosa cell-to-cell signaling is required for virulence in a model of acute pulmonary infection

Cell-to-cell signaling controls many virulence genes in Pseudomonas aeruginosa. We tested the virulence of las and rhl quorum-sensing mutants in neonatal mice. A lasI rhlI double mutant was nearly avirulent, and the respective single mutant strains were reduced in virulence compared with the wild-type strain. Quorum sensing plays a role in P. aeruginosa pneumonia in neonatal mice.     

Reovirus-like agent (rotavirus) from lambs.

Rotavirus particles were demonstrated by electron microscopy in the feces of lambs with diarrhea. Rotavirus antigen was synthesized in cell cultures infected with filtrates of the diarrheic feces, but the virus was not adapted to grow serially in cell cultures. An antigenic relationship between rotaviruses from lambs, pigs, and calves was demonstrated by immunofluorescence. Colostrum-deprived lambs were infected with the lamb rotavirus, and the virus was passaged in lambs. Viral replication occurred in the villous epithelial cells of the small intestine, and the virus was excreted in the feces up to 78 h postinfection. Diarrhea was not observed in the experimentally infected lambs.     

GDF-8 propeptide binds to GDF-8 and antagonizes biological activity by inhibiting GDF-8 receptor binding

GDF-8 is a new member of the TGF-beta superfamily which appears to be a negative regulator of skeletal muscle mass. Factors which regulate the biological activity of GDF-8 have not yet been identified. However, the biological activities of TGF-beta superfamily members, TGF-beta1, -beta2 and -beta3, can be inhibited by noncovalent association with TGF-beta1, -beta2 and beta3 propeptides cleaved from the amino-termini of the TGF-beta precursor proteins. In contrast, the propeptides of other TGF-beta superfamily members do not appear to be inhibitory. In this investigation, we demonstrate that purified recombinant GDF-8 propeptide associates with purified recombinant GDF-8 to form a noncovalent complex, as evidenced by size exclusion chromatography and chemical crosslinking analysis. Furthermore, we show that GDF-8 propeptide inhibits the biological activity of GDF-8 assayed on A204 rhabdomyosarcoma cells transfected with a (CAGA)12 reporter construct. Finally, we demonstrate that GDF-8 propeptide inhibits specific GDF-8 binding to L6 myoblast cells. Collectively, these data identify the GDF-8 propeptide as an inhibitor of GDF-8 biological activity.     

CONSISTENCY OF PERFORMANCE CHANGE AND AUTONOMIC RESPONSE AS A FUNCTION OF EXPRESSED ATTITUDE TOWARD A SPECIFIC STRESS SITUATION

A persistent problem in stress research has been that some individuals may show impairment, while others show improvement or no change in performance under stress. Attempts to relate this variance in performance to general anxiety or other personality variables have generally not been too successful. Based upon responses to a fear of shock item in an attitude questionnaire, Ss were classified as “high fear of shock” or “low fear of shock” types. Half of the Ss in each group were assigned a perceptual-motor task; the others were assigned a cognitive-interference task. After training, all Ss were informed that they would be required to maintain their training performance levels in a situation in which they would be shocked if performance declined. Performance and heart rate measures taken during training were compared with the same measures taken under the threat-of-shock conditions. Results indicate significant differences between groups in both performance and physiological activity with “high fear of shock”Ss exhibiting relatively greater performance impairment and increased heart rate.     

Fictive motor patterns in chronic spinal cats.

1. Fictive motor patterns were recorded in hind leg nerves of 10 adult chronic spinal cats (spinalized at T13). Four of these animals had been trained to step with their hind legs on a treadmill (late-spinal animals), whereas the remainder received no training and were examined a short time after spinalization (early-spinal animals). 2. A fictive pattern resembling the locomotor pattern for stepping was evoked in all animals in response to stimulation of the skin of the perineal region. (2-[2,6-Dichloroaniline]-2-imidazoline) hydrochloride (Clonidine) at doses ranging from 100 to 500 micrograms/kg iv facilitated the production of this pattern, particularly in early-spinal animals. 3. The fictive locomotor pattern in late-spinal animals was more complex than that occurring in early-spinal animals. In the latter the pattern consisted of an alternation of activity in flexor and extensor nerves, and changing leg position did not qualitatively alter the pattern, whereas in late-spinal animals the relative durations of the bursts in different flexors were usually not the same, and the pattern of flexor activity was dependent on leg position. 4. Moving the legs from extension to flexion progressively decreased the duration of flexor bursts, increased the cycle period, and decreased the ease with which the pattern could be evoked in both early- and late-spinal animals. 5. 1-beta-3,4-Dihydroxyphenylalanine (DOPA)/Isonocotinic acid 2-[(2-benzylcarbamoyl)ethyl]hydrazide (Nialamide) treatment following Clonidine in early-spinal animals increased the complexity of flexor burst activity. This, and other observations, indicates that DOPA and Clonidine do not have strictly identical actions on the locomotor pattern generator. 6. Stimulation of the paws in late-spinal animals produced two patterns of activity distinctly different from the locomotor pattern. of activity distinctly different from the locomotor pattern. One was a short sequence of high-frequency rhythmic activity (at approximately 8 c/s) in response to gently stimulating one paw with a water jet, and the other was a slow rhythm in flexor nerves in response to squeezing the paw. 7. The main conclusion of this investigation is that three distinctly different fictive motor patterns can be generated in chronic spinal cats depending on the method and site of stimulation. These patterns correspond to three different behaviors (locomotion, paw shake, and rhythmic leg flexion) that can be elicited in behaving chronic spinal cats in response to the same stimuli.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of ω-conotoxin-sensitive Ca2+ channel currents by internal Mg2+ in cultured rat cerebellar granule neurones

The effects of changing the intracellular concentrations of either free Mg²⁺ ions ([Mg²⁺]_i) or Mg²⁺-bound adenosine triphosphate ([Mg · ATP]_i) on Ca²⁺ channel currents were assessed in cultured rat cerebellar granule neurones using the whole-cell patch-clamp technique. Raising [Mg²⁺]_i from 0.06 mM to 1.0 mM inhibited Ca²⁺ channel currents by approximately 50%. The action of -conotoxin GVIA (-CgTX), a selective inhibitor of N-type Ca²⁺ channels was also investigated. With increasing [Mg²⁺]_i, the proportion of current irreversibly blocked by -CgTX was reduced, and was negligible (approximately 5 pA of current) in the presence of [Mg²⁺]_i values of 0.5 mM or greater. Block of the -CgTX-sensitive current accounted for the reduction in total current by concentrations of [Mg²⁺]_i to 0.5 mM. Raising [Mg²⁺]_i had no effect on the rate of decay of Ca²⁺ currents, but did produce a negative shift in current activation, possibly due to a non-specific interaction with negative surface charge. Altering [Mg · ATP]_i from 0.3 to 5.0 mM caused a twofold increase in the size of currents without affecting the proportion of current sensitive to -CgTX. [Mg²⁺]_i was also effective in inhibiting the Ca²⁺ channel current following potentiation by increasing [Mg · ATP]_i. These data suggest that -CgTX-sensitive current in these cells is selectively inhibited by internal Mg²⁺ whereas both -CgTX-sensitive and -resistant components of current are potentiated by internal Mg · ATP. The mechanism by which Mg²⁺ inhibits N-type channels is unclear, but may involve an open channel block.