Functional and physical coupling of voltage-sensitive calcium channels with exocytotic proteins: ramifications for the secretion mechanism

The secretion of neurotransmitters is a rapid Ca(2+)-regulated process that brings about vesicle fusion with the plasma membrane. This rapid process (< 100 microseconds) involves multiple proteins located at the plasma and vesicular membranes. Because of their homology to proteins participating in constitutive secretion and protein trafficking, they have been characterized extensively. The sequential events that lead these proteins to vesicle docking and fusion are still unclear. We will review recent studies that demonstrate the operative role played by voltage-sensitive Ca(2+) channels and discuss the relevance for the process of evoked transmitter release. The regulation of Ca(2+) influx by syntaxin, synaptosome-associated protein of 25 kDa (SNAP-25) and synaptotagmin, and the reciprocity of these proteins in controlling the kinetic properties of the channel will be discussed. Calcium channel and synaptic proteins expressed in Xenopus oocytes demonstrate a strong functional interaction, which could be pertinent to the mechanism of secretion. First, the voltage-sensitive Ca(2+) channels are negatively modulated by syntaxin: this inhibition is reversed by synaptotagmin. Second, the modulation of N-type Ca(2+) channel activation kinetics strongly suggests that the vesicle could be docked at the plasma membrane through direct interaction with synaptotagmin. Finally, these interactions provide evidence for the assembly of the voltage-sensitive Ca(2+) channel with syntaxin 1A, SNAP-25 and synaptotagmin into an excitosome complex: a putative fusion complex with a potential role in the final stages of secretion. Studies suggest that cross-talk between the synaptic proteins and the channel in a tightly organized complex may enable a rapid secretory response to an incoming signal such as membrane depolarization.     

Biodefense research: an emerging conundrum

Biodefense research creates a conundrum for the scientific community--what are the permissible limits of biodefense research and how can knowledge in the life sciences be protected against misuse? As biodefense research expands to counter the threat of bioterrorism, so does suspicion and the need for guidance to respond to these concerns.     

The C2A domain of synaptotagmin alters the kinetics of voltage-gated Ca2+ channels Ca(v)1.2 (Lc-type) and Ca(v)2.3 (R-type)

Biochemical and genetic studies implicate synaptotagmin (Syt 1) as a Ca2+ sensor for neuronal and neuroendocrine neurosecretion. Calcium binding to Syt 1 occurs through two cytoplasmic repeats termed the C2A and C2B domains. In addition, the C2A domain of Syt 1 has calcium-independent properties required for neurotransmitter release. For example, mutation of a polylysine motif (residues 189-192) reverses the inhibitory effect of injected recombinant Syt 1 C2A fragment on neurotransmitter release from PC12 cells. Here we examined the requirement of the C2A polylysine motif for Syt 1 interaction with the cardiac Cav1.2 (L-type) and the neuronal Cav2.3 (R-type) voltage-gated Ca2+ channels, two channels required for neurotransmission. We find that the C2A polylysine motif presents a critical interaction surface with Cav1.2 and Cav2.3 since truncated Syt 1 containing a mutated motif (Syt 1*1-264) was ineffective at modifying the channel kinetics. Mutating the polylysine motif also abolished C2A binding to Lc753-893, the cytosolic interacting domain of Syt 1 at Cav1.2 1 subunit. Syt 1 and Syt 1* harboring the mutation at the KKKK motif modified channel activation, while Syt 1* only partially reversed the syntaxin 1A effects on channel activity. This mutation would interfere with the assembly of Syt 1/channel/syntaxin into an exocytotic unit. The functional interaction of the C2A polylysine domain with Cav1.2 and Cav2.3 is consistent with tethering of the secretory vesicle to the Ca2+ channel. It indicates that calcium-independent properties of Syt 1 regulate voltage-gated Ca2+ channels and contribute to the molecular events underlying transmitter release.     

Recovery of DNA from soils and sediments

Experiments were performed to evaluate the effectiveness of two different methodological approaches for recovering DNA from soil and sediment bacterial communities: cell extraction followed by lysis and DNA recovery (cell extraction method) versus direct cell lysis and alkaline extraction to recover DNA (direct lysis method). Efficiency of DNA recovery by each method was determined by spectrophotometric absorbance and using a tritiated thymidine tracer. With both procedures, the use of polyvinylpolypyrrolidone was important for the removal of humic compounds to improve the purity of the recovered DNA; without extensive purification, various restriction enzymes failed to cut added target DNA. Milligram quantities of high-purity DNA were recovered from 100-g samples of both soils and sediments by the direct lysis method, which was a greater than 1-order-of-magnitude-higher yield than by the cell extraction method. The ratio of labeled thymidine to total DNA, however, was higher in the DNA recovered by the cell extraction method. than by the direct lysis method, suggesting that the DNA recovered by the cell extraction method came primarily from active bacterial cells, whereas that recovered by the direct lysis method may have contained DNA from other sources.     

Model suicide vector for containment of genetically engineered microorganisms.

A model suicide vector (pBAP19h), designed for the potential containment of genetically engineered microorganisms, was made by constructing a plasmid with the hok gene, which codes for a lethal polypeptide, under the control of the lac promoter. The vector plasmid also codes for carbenicillin resistance. In the absence of carbenicillin, induction of the hok gene in vitro caused elimination of all detectable cells containing the suicide vector; pBAP19h-free cells of the culture survived and grew exponentially. In the presence of carbenicillin, however, the number of cells containing pBAP19h initially declined after induction of hok but then multiplied exponentially. The surviving cells still had a fully functional hok gene and had apparently developed resistance to the action of the Hok polypeptide. Thus, high selective pressure against the loss of the suicide vector led to a failure of the system. Soil microcosm experiments confirmed the ability of a suicide vector to restrict the growth of a genetically engineered microorganism in the absence of selective pressure against the loss of the plasmid, with 90 to 99% elimination of hok-bearing cells within 24 h of hok induction. However, some pBAP19h-bearing cells survived in the soil microcosms after hok induction. The surviving cells contained an active hok gene but were not capable of normal growth even after elimination of the hok gene; it appears that a mutation that made them Hok resistant also reduced their capacity for membrane functions needed for energy generation and exponential cell growth. Thus, the model suicide vector was shown to be functional in soil as well as in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of coliform bacteria and Escherichia coli by multiplex polymerase chain reaction: comparison with defined substrate and plating methods for water quality monitoring.

Multiplex polymerase chain reaction (PCR) and gene probe detection of target lacZ and uidA genes were used to detect total coliform bacteria and Escherichia coli, respectively, for determining water quality. In tests of environmental water samples, the lacZ PCR method gave results statistically equivalent to those of the plate count and defined substrate methods accepted by the U.S. Environmental Protection Agency for water quality monitoring and the uidA PCR method was more sensitive than 4-methylumbelliferyl-beta-D-glucuronide-based defined substrate tests for specific detection of E. coli.     

Detection of Giardia cysts by using the polymerase chain reaction and distinguishing live from dead cysts.

A method was developed for the detection of Giardia cysts by using the polymerase chain reaction (PCR) and the giardin gene as the target. DNA amplification by PCR, using giardin DNA as the target, resulted in detection of both live and dead cysts. When giardin mRNA was used as the target, the ability to amplify cDNA by PCR depended on the mode of killing. Cysts killed by freezing were not detected by PCR when giardin mRNA was the target. Cysts killed by heating or exposure to monochloramine, however, gave positive detection signals for both DNA and giardin mRNA targets. The amount of giardin mRNA and total RNA was significantly increased in live cysts following the induction of excystation. Cysts killed by freezing, heating, or exposure to monochloramine did not show a change in RNA content. The detection of the giardin gene by PCR permits a sensitive and specific diagnosis for Giardia spp. Discrimination between live and dead cysts can be made by measuring the amounts of RNA or PCR-amplified product from the giardin mRNA target before and after the induction of excystation.     

Direct effect of angiotensin II on in-vitro perfused rabbit ovary

The effects of angiotensin II (AII) and its receptor blocker, saralasin (SAR), on ovulation and oocyte maturation were investigated in an isolated, in-vitro perfused rabbit ovary. Ovulation and oocyte maturation were induced by AII in the absence of human chorionic gonadotrophin (hCG). SAR inhibited ovulation induced by AII or hCG, but not oocyte maturation. AII appears to play a critical role in follicle rupture, but not in resumption of oocyte meiosis.     

Maintenance and killing efficiency of conditional lethal constructs inPseudomonas putida

Summary Conditional lethal (suicidal) genetic constructs were designed and employed in strains of Pseudomonads as models for containment of geneticallyengineerd microbes that may be deliberately released into the environment. A strain ofPseudomonas putida was formed with a suicide vector designated pBAP24h that was constructed by cloning the host killing gene (hok) into the RSF1010 plasmid pVDtac24 and placing it under the control of thetac promoter. Afterhok induction inP. putida only 40% of surviving cells continued to bear thehok sequences within 4 h of induction; in contrast, 100% of the cells in uninduced controls borehok. A few survivors that demonstrated resistance tohok-induced killing developed inP. putida, which may have been due to a mutation or physiological adaptation that rendered the membrane resistant tohok. Conditional lethal strains ofP. putida also were formed by insertinggef (a chromosomal homolog ofhok) under the control of thetac promoter into the chromosome using a transposon. Constructs with chromosomalgef, as well as an RK2-derived plasmid construct containinggef, were only marginally more stable than thehok constructs; they were effective in killingP. putida when induced and within 2 h post-induction killing from eithergef construct resulted in a 10³–10⁵-fold reduction in viable cell count compared to uninduced controls.