Early detection of negative BACTEC MGIT 960 cultures by PCR-reverse cross-blot hybridization assay

We evaluated the efficacy of a PCR-reverse cross-blot hybridization assay, a test which permits identification of mycobacteria by means of species-specific probes and a Mycobacterium-specific probe, for early detection of negative BACTEC MGIT 960 mycobacterial cultures. Aliquots of 549 cultures were collected 7 days after the culture media were inoculated with various clinical specimens and tested with the molecular assay. PCR results were compared to those obtained at the end times with the BACTEC MGIT 960 system. Of the 549 specimens analyzed, 484 were found to be negative and 64 were found positive by both methods; one specimen, found to be positive by the BACTEC MGIT 960 system, was identified as negative by the molecular assay. In view of its high negative predictive value (99.8%), the PCR-reverse cross-blot hybridization assay appears to be a valid tool for early detection of negative BACTEC MGIT 960 cultures.     

Isolation of a new clathrin heavy chain gene with muscle-specific expression from the region commonly deleted in velo-cardio-facial syndrome

Velo-cardio-facial syndrome (VCFS) and DiGeorge syndrome (DGS) are developmental disorders characterized by a spectrum of phenotypes including velopharyngeal insufficiency, conotruncal heart defects and facial dysmorphology among others. Eighty to eighty-five percent of VCFS/DGS patients are hemizygous for a portion of chromosome 22. It is likely that the genes encoded by this region play a role in the etiology of the phenotypes associated with the disorders. Using a cDNA selection protocol, we isolated a novel clathrin heavy chain cDNA (CLTD) from the VCFS/DGS minimally deleted interval. The cDNA encodes a protein of 1638 amino acids. CLTD shares significant homology, but is not identical to the ubiquitously expressed clathrin heavy chain gene. The CLTD gene also shows a unique pattern of expression, having its maximal level of expression in skeletal muscle. Velopharyngeal insufficiency and muscle weakness are common features of VCFS patients. Based on the location and expression pattern of CLTD, we suggest hemizygosity at this locus may play a role in the etiology of one of the VCFS-associated phenotypes.      

Formaldehyde in cryoprotectant propanediol and effect on mouse zygotes

Cryopreservation of human zygotes and embryos has been routinely performed by in-vitro fertilization clinics for many years. Karran and Legge (1996) first reported that formaldehyde (FA) present in the cryoprotective solutions can have a deleterious effect on mouse oocytes. FA is a cytotoxic, carcinogenic and mutagenic chemical. The effect of FA on mouse zygotes was investigated. In addition, the concentrations of FA in propanediol (PROH) obtained from various sources were determined. Pooled 1-cell embryos were dispensed into droplets of modified Ham's F10 or human tubal fluid containing various concentrations of FA. Since bovine serum albumin (BSA) may minimize toxicity additional trials were done as above in the absence of BSA. FA concentration in the standard 1.5 M PROH, from different sources in water, was measured in the same assay using a standard curve of 0-100 microM FA. FA in a complex medium had a significant deleterious effect on embryo development and hatching but only at 1 mM concentration (P < 0.000001; see Tables I-III). There was no significant effect of FA at 100 microM. However, in a simple medium even 50 microM FA decreased embryo hatching. FA was present in 1.5 M PROH from different sources (range 1.0-35.3 microM concentration). It appears that FA concentrations do not increase with storage because FA concentrations were low even after opening and storage for 3 years on the shelf. This suggests that FA is a contaminant during the manufacturing process and may vary from manufacturer to manufacturer and batch to batch. Until further studies are done to confirm the lack of toxicity to embryos during cryopreservation (with or without FA scavengers) it may be prudent to screen all batches of cryoprotectants for FA as part of quality control.      

Altered gating of HERG potassium channels by cobalt and lanthanum

Activation of the rapid, delayed rectifier K current (IKr) is important for normal repolarization of cardiac action potentials, especially in mammalian ventricular muscle. The study of this current has been greatly aided by the discovery that the human ether-a-go-go-related gene (HERG) encodes the pore-forming alpha subunits of these channels. As for other voltage-activated K+ channels, divalent and trivalent cations affect the gating of HERG channels by screening negative membrane surface charges or by direct interaction with the channel gating mechanism. Previous studies have reported that IKr of myocytes, and HERG channels heterologously expressed in Xenopus oocytes, are reduced by external Co2+ and La3+. We have reinvestigated the "blocking" effect of Co2+ and La3+ on HERG channels expressed in Xenopus oocytes. At concentrations previously reported to block IKr or HERG current (IHERG), Co2+ (10 mM) and La3+ (10 microM) had only small effects on the magnitude of fully activated IHERG. The apparent block results from altered kinetics and voltage dependence of gating, similar to the effects of Ca2+ on HERG channels. Under control conditions, the half-points for voltage-dependent activation and inactivation of HERG were -35+/-2.1 and -76.3+/-1.7 mV, respectively. Co2+ and La3+ accelerated the rate of deactivation, decreased the rate of current activation, and shifted the half-point of the HERG channel activation curve by +53 and +65 mV, respectively. Co2+ shifted the voltage dependence of inactivation by + 14 mV, whereas La3+ had no effect. Co2+ also slowed the onset of IHERG inactivation and accelerated the rate of recovery from inactivation. These results indicate that reduction of IHERG by Co2+ (10 mM) and La3+ (10 microM) during depolarizing pulses is caused by a positive shift in the voltage dependence of activation, and does not result from pore block.     

Novel KChIP2 isoforms increase functional diversity of transient outward potassium currents

Kv4.3 channels conduct transient outward K⁺ currents in the human heart and brain where they mediate the early phase of action potential repolarization. KChIP2 proteins are members of a new class of calcium sensors that modulate the surface expression and biophysical properties of Kv4 K⁺ channels. Here we describe three novel isoforms of KChIP2 with an alternatively spliced C-terminus (KChIP2e, KChIP2f) or N-terminus (KChIP2g). KChIP2e and KChIP2f are expressed in the human atrium, whereas KChIP2g is predominantly expressed in the brain. The KChIP2 isoforms were coexpressed with Kv4.3 channels in Xenopus oocytes and currents recorded with two-microelectrode voltage-clamp techniques. KChIP2e caused a reduction in current amplitude, an acceleration of inactivation and a slowing of the recovery from inactivation of Kv4.3 currents. KChIP2f increased the current amplitude and slowed the rate of inactivation, but did not alter the recovery from inactivation or the voltage of half-maximal inactivation of Kv4.3 channels. KChIP2g increased current amplitudes, slowed the rate of inactivation and shifted the voltage of half-maximal inactivation to more negative potentials. The biophysical changes induced by these alternatively spliced KChIP2 proteins differ markedly from previously described KChIP2 proteins and would be expected to increase the diversity of native transient outward K⁺ currents.