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Potential insulin secretagogue properties of an acetoxymethyl ester of a cAMP analog (8-pCPT-2'-O-Me-cAMP-AM) that activates the guanine nucleotide exchange factors Epac1 and Epac2 were assessed using isolated human islets of Langerhans. RT-QPCR demonstrated that the predominant variant of Epac expressed in human islets was Epac2, although Epac1 was detectable. Under conditions of islet perifusion, 8-pCPT-2'-O-Me-cAMP-AM (10 [micro]M) potentiated first-and second-phase 10 mM glucose-stimulated insulin secretion (GSIS) while failing to influence insulin secretion measured in the presence of 3 mM glucose. The insulin secretagogue action of 8-pCPT-2'-O-Me-cAMP-AM was associated with depolarization and an increase of [[[Ca.sup.2+]].sub.i] that reflected both [Ca.sup.2+] influx and intracellular [Ca.sup.2+] mobilization in islet [beta]-cells. As expected for an Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP-AM (10 [micro]M) failed to stimulate phosphorylation of PKA substrates CREB and Kemptide in human islets. Furthermore, 8-pCPT-2'-O-Me-cAMP-AM (10 [micro]M) had no significant ability to activate AKAR3, a PKA-regulated biosensor expressed in human islet cells by viral transduction. Unexpectedly, treatment of human islets with an inhibitor of PKA activity (H-89) or treatment with a cAMP antagonist that blocks PKA activation (Rp-8-CPT-cAMPS) nearly abolished the action of 8-pCPT-2'-O-Me-cAMP-AM to potentiate GSIS. It is concluded that there exists a permissive role for PKA activity in support of human islet insulin secretion that is both glucose dependent and Epac regulated. This permissive action of PKA may be operative at the insulin secretory granule recruitment, priming, and/or postpriming steps of [Ca.sup.2+]-dependent exocytosis. protein kinase A; exocytosis; adenosine-3',5'-cyclic monophosphate; exchange protein directly activated by adenosine-3',5'-cyclic monophosphate; calcium doi:10.1152/ajpendo.00630.2009.

The oral glucose tolerance test and meal tolerance test are common clinical tests of the glucose-insulin system. Several mathematical models have been suggested as means to extract information about [beta]-cell function from data from oral tolerance tests. Any such model needs to be fairly simple but should at the same time be linked to the underlying biology of the insulin-secreting [beta]-cells. The scope of the present work is to present a way to make such a connection using a recent model describing intracellular mechanisms. We show how the three main components of oral minimal secretion models, derivative control, proportional control, and delay, are related to subcellular events, thus providing mechanistic underpinning of the assumptions of the minimal models. secretory granules; [beta]-cell function; C-peptide secretion; threshold distribution; mathematical models doi:10.1152/ajpendo.00670.2009

Rates of liquid secretion and absorption across the bronchopulmonary airways are important for regulating airway surface liquid volume and maintaining mucociliary transport. The current study demonstrates the feasibility of measuring not just net liquid movements but unidirectional liquid movements across isolated intact bronchi from swine. Airways were liquid filled to assess both net liquid movements, and, in the presence of NPPB to selectively inhibit secretion, unidirectional absorption. Unidirectional liquid secretion rates were determined by subtraction. For comparison, net liquid movements were assessed in air-filled airways in parallel. In the absence of secretagogues, unidirectional absorption was observed (4.63 [+ or -] 0.53 [micro]l*[cm.sup.-2]*[h.sup.-1]) with little unidirectional secretion (1.42 [+ or -] 0.36 [micro]*[cm.sup.-2]*[h.sup.-1]). ACh, substance P (SP), and vasoactive intestinal peptide (VIP) all induced unidirectional secretion (10.64 [+ or -] 1.52 [micro]l*[cm.sup.-2]*[h.sup.-1], 14.16 [+ or -] 1.39 [micro]l*[cm.sup.-2]*[h.sup.-1], and 4.25 [+ or -] 0.25 [micro]l*[cm.sup.-2]*[h.sup.-1], respectively) without affecting unidirectional absorption. Net liquid secretion in air-filled airways was close to that in liquid-filled airways except with VIP. VIP induced net secretion in air-filled airways (4.44 [+ or -] 1.26 [micro]l*[cm.sup.-2]*[h.sup.-1]), but negligible net change in liquid movement occurred in liquid-filled airways. This effect was likely to have been caused by the higher solid content of the VIP-induced mucous liquid (3.98 [+ or -] 0.26%) compared with the ACh- and SP-induced liquid (2.06 [+ or -] 0.07% and 2.15 [+ or -] 0.07%, respectively). We conclude that this technique allows important quantitative distinctions to be made between liquid secretion and absorption in intact bronchial airways. liquid transport; secretion; absorption; airway epithelia; submucosal glands doi: 10.1152/ajplung.00253.2009

Type III secretion (T3S) functions in establishing infections in a large number of Gram-negative bacteria, yet little is known about how host cell properties might function in this process. We used the opportunistic pathogen Pseudomonas aeruginosa and the ability to alter host cell sensitivity to Pseudomonas T3S to explore this problem. HT-29 epithelial cells were used to study cellular changes associated with loss of T3S sensitivity, which could be induced by treatment with methyl-beta-cyclodextrin or perfringolysin O. HL-60 promyelocytic cells are innately resistant to Pseudomonas T3S and were used to study cellular changes occurring in response to induction of T3S sensitivity, which occurred following treatment with phorbol esters. Using both cell models, a positive correlation was observed between eukaryotic cell adherence to tissue culture wells and T3S sensitivity. In examining the type of adhesion process linked to T3S sensitivity in HT-29 cells, a hierarchical order of protein involvement was identified that paralleled the architecture of leading edge (LE) focal complexes. Conversely, in HL-60 cells, induction of T3S sensitivity coincided with the onset of LE properties and the development of actin-rich projections associated with polarized cell migration. When LE architecture was examined by immunofluorescent staining for actin, Rac1, IQ-motif-containing GTPase-activating protein 1 (IQGAP1)and phosphatidylinositol 3 kinase (PI3 kinase), intact LE structure was found to closely correlate with host cell sensitivity to P. aeruginosa T3S. Our model for host cell involvement in Pseudomonas T3S proposes that cortical actin polymerization at the LE alters membrane properties to favour T3S translocon function and the establishment of infections, which is consistent with Pseudomonas infections targeting wounded epithelial barriers undergoing cell migration. DOI 10.1099/mic.0.033241-0

The type III secretion system (T3SS) encoded by Salmonella pathogenicity island 1 (SPI-1) is important for the invasion of epithelial cells during development of Salmonella-associated enterocolitis. It has been suggested that the level and timing of the expression of the SPI-1 T3SS proteins and effectors dictate the consequences of bacterial infection and pathogenesis. However, the expression of these proteins has not been extensively studied in vivo, especially during the later stages of salmonellosis when the infection is established. We have constructed recombinant Salmonella strains that contain a FLAG epitope inserted in-frame to genes invJ, prgJ, sipC, sipD, sopA and sopB, and investigated the expression of the tagged proteins both in vitro and in vivo during murine salmonellosis. Mice were inoculated intraperitoneally or intragastrically with the tagged Salmonella strains. At different time points post-infection, bacteria were recovered from various organs, and the expression of the tagged proteins was determined. Our results provide direct evidence that PrgJ and SipD are expressed in Salmonella colonizing the liver and ileum of infected animals at both the early and late stages of infection. Furthermore, our study has shown that the InvJ protein is expressed preferentially in Salmonella colonizing the ileum but not the liver, while SipC is expressed preferentially in Salmonella colonizing the liver but not the ileum. Thus, Salmonella appears to express different SPI-1 proteins and effectors when colonizing specific tissues. Our results suggest that differential expression of these proteins may be important for tissue-specific aspects of bacterial pathogenesis such as gastroenterititis in the ileum and systemic infection in the liver. DOI 10.1099/mic.0.032318-0

Under conditions of nitrogen stress, leguminous plants form symbioses with soil bacteria called rhizobia. This partnership results in the development of structures called root nodules, in which differentiated endosymbiotic bacteria reduce molecular dinitrogen for the host. The establishment of rhizobium-legume symbioses requires the bacterial synthesis of oligosaccharides, exopolysaccharides, and capsular polysaccharides. Previous studies suggested that the 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo) homopolymeric capsular polysaccharide produced by strain Sinorhizobium meliloti Rm1021 contributes to symbiosis with Medicago sativa under some conditions. However, a conclusive symbiotic role for this polysaccharide could not be determined due to a lack of mutants affecting its synthesis. In this study, we have further characterized the synthesis, secretion, and symbiotic function of the Kdo homopolymeric capsule. We showed that mutants lacking the enigmatic rkp-1 gene cluster fail to display the Kdo capsule on the cell surface but accumulate an intracellular polysaccharide of unusually high [M.sub.r]. In addition, we have demonstrated that mutations in kdsB2, smb20804, and smb20805 affect the polymerization of the Kdo homopolymeric capsule. Our studies also suggest a role for the capsular polysaccharide in symbiosis. Previous reports have shown that the overexpression of rkpZ from strain Rm41 allows for the symbiosis of exoY mutants of Rm1021 that are unable to produce the exopolysaccharide succinoglycan. Our results demonstrate that mutations in the rkp-1 cluster prevent this phenotypic suppression of exoY mutants, although mutations in kdsB2, smb20804, and smb20805 have no effect. doi: 10.1128/JB.00466-09

Recently, a novel type of secretory pathway, type VII secretion systems (T7SSs), has been characterized in mycobacteria. The chromosomes of Mycobacterium tuberculosis and Mycobacterium bovis encode five T7SSs (ESX-1 to ESX-5). The best characterized of them, ESX-1, is involved in host-pathogen interactions, and its deletion is one of the main causes of M. bovis BCG attenuation. Another T7SS, ESX-3, has been previously shown to be transcriptionally controlled by the zinc uptake repressor (Zur) and by the iron-dependent transcriptional repressor (IdeR), suggesting that it might be involved in zinc and iron homeostasis. In this study, we characterized an M. tuberculosis conditional mutant in which transcription of the ESX-3 gene cluster can be downregulated by anhydrotetracycline. We showed that this T7SS is essential for growth and that this phenotype can be complemented by zinc, iron, or supernatant from a wild-type parental strain culture, demonstrating that the ESX-3 secretion system is responsible for the secretion of some soluble factor(s) required for growth that is probably involved in optimal iron and zinc uptake. doi: 10.1128/JB.00756-09

Secretory compartments of neurons, endocrine cells, and exocrine glands are acidic and contain high levels of labile [Zn.sup.2+]. Previously, we reported evidence that acidity is regulated, in part, by the content of [Zn.sup.2+] in the secretory [i.e., tubulovesicle (TV)] compartment of the acid-secreting gastric parietal cell. Here we report studies focusing on the mechanisms of [Zn.sup.2+] transport by the TV compartment in the mammalian (rabbit) gastric parietal cell. Uptake of [Zn.sup.2+] by isolated TV structures was monitored with a novel application of the fluorescent [Zn.sup.2+] reporter N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ). Uptake was suppressed by removal of external ATP or blockade of [H.sup.+]-[K.sup.+]-ATPase that mediates luminal acid secretion. Uptake was diminished with dissipation of the proton gradient across the TV membrane, suggesting [Zn.sup.2+]/[H.sup.+] antiport as the connection between [Zn.sup.2+] uptake and acidity in the TV lumen. In isolated gastric glands loaded with the reporter fluozin-3, inhibition of [H.sup.+]-[K.sup.+]-ATPase arrested the flow of [Zn.sup.2+] from the cytoplasm to the TV compartment and secretory stimulation with forskolin enhanced vectorial movement of cytoplasmic [Zn.sup.2+] into the tubulovesicle/lumen (TV/L) compartment. Our findings suggest that [Zn.sup.2+] accumulation in the TV/L compartment is physiologically coupled to secretion of acid. These findings offer novel insight into mechanisms regulating [Zn.sup.2+] homeostasis in the gastric parietal cell and potentially other cells in which acidic subcellular compartments serve signature functional roles. gastric gland; tubulovesicles; zinc; acidity regulation doi: 10.1152/ajpcell.00577.2008

Cells of wild-type Yersinia pestis exhibit a low-calcium response (LCR) defined as bacteriostasis with expression of a pCD-encoded type III secretion system (T3SS) during cultivation at 37 [degrees]C without added [Ca.sup.2+] versus vegetative growth with downregulation of the T3SS with [Ca.sup.2+] ([greater than or equal to]2.5 mM). Bacteriostasis is known to reflect cumulative toxicity of Na+, L-glutamic acid and culture pH; control of these variables enables full-scale growth ('rescue') in the absence of [Ca.sup.2+]. Several T3SS regulatory proteins modulate the LCR, because their absence promotes a [Ca.sup.2+]-blind phenotype in which growth at 37 [degrees]C ceases and the T3SS is constitutive even with added [Ca.sup.2+]. This study analysed the connection between the LCR and [Ca.sup.2+] by determining the response of selected [Ca.sup.2+]-blind mutants grown in [Ca.sup.2+]-deficient rescue media containing Na+ plus L-glutamate (pH 5.5), where the T3SS is not expressed, L-glutamate alone (pH 6.5), where L-aspartate is fully catabolized, and Na+ alone (pH 9.0), where the electrogenic sodium pump NADH : ubiquinone oxidoreductase becomes activated. All three conditions supported essentially full-scale [Ca.sup.2+]-independent growth at 37 [degrees]C of wild-type Y. pestis as well as IcrG and yopN mutants (possessing a complete but dysregulated T3SS), indicating that bacteriostasis reflects a Na+-dependent lesion in bioenergetics. In contrast, mutants lacking the negative regulator YopD or the YopD chaperone (LcrH) failed to grow in any rescue medium and are therefore truly temperature-sensitive. The [Ca.sup.2+]-blind yopD phenotype was fully suppressed in a [Ca.sup.2+]-independent background lacking the injectisome-associated inner-membrane component YscV but not peripheral YscK, suggesting that the core translocon energizes YopD .

In Escherichia coli, the assembly of outer-membrane proteins (OMP) requires the BAM complex and periplasmic chaperones, such as SurA or DegP. Previous work has suggested a potential link between OMP assembly and expression of the genes encoding type-III secretion systems. In order to test this hypothesis, we studied the role of the different lipoproteins of the BAM complex (i.e. BamB, BamC, BamD and BamE), and the periplasmic chaperones SurA and DegP, in these two phenotypes in Salmonella. Analysis of the corresponding deletion mutants showed that, as previously described with the [DELTA]bamB mutant, BamD, SurA and, to a lesser extent, BamE play a role in outer-membrane biogenesis in Salmonella Enteritidis, while the membrane was not notably disturbed in [DELTA]bamC and [DELTA]degP mutants. Interestingly, we found that BamD is not essential in Salmonella, unlike its homologues in Escherichia coil and Neisseria gonorrhoeae. In contrast, BamD was the only protein required for full expression of T3SS-1 and flagella, as demonstrated by transcriptional analysis of the genes involved in the biosynthesis of these T3SSs. In line with this finding, bamD mutants showed a reduced secretion of effector proteins by these T3SSs, and a reduced ability to invade HT-29 cells. As [DELTA]surA and [DELTA]bamE mutants had lower levels of OMPs in their outer membrane, but showed no alteration in T3SS-1 and flagella expression, these results demonstrate the absence of a systematic link between an OMP assembly defect and the downregulation of T3SSs in Salmonella; therefore, this link appears to be related to a more specific mechanism that involves at least BamB and BamD.

Neisseria meningitidis, an obligate human pathogen, remains a leading cause of meningitis and fatal sepsis. Meningococci are known to secrete a family of proteins, such as FrpC, with sequence similarity to the repeat-in-toxin (RTX) proteins via the type I secretion system. The meningococcal type I secretion proteins are encoded at two distant genetic loci, NMB1400 (hlyB) and NMB173811737 (hlyD/tolC), and are separated from the RTX toxin-like substrates. We have characterized the promoter elements of both hlyB and hlyD by primer extension and lacZ reporter fusions and revealed the growth phase-dependent upregulation of both genes. In addition, we showed that the MisR/MisS two-component system negatively regulates the expression of hlyB and hlyD/tolC. Direct binding of MisR to hlyB and hlyD promoters was demonstrated by electrophoretic mobility shift assay (EMSA), and DNase I protection assays identified MisR binding sites overlapping the promoter elements. Direct repression of hlyB transcription by MisR was supported by in vitro transcription assays. Mutations in the MisR/S system affected, but did not eliminate, the growth phase-dependent upregulation of hlyB, suggesting additional regulatory mechanisms. Increased secretion of RTX toxin-like proteins was detected in the cell-free media from misS mutant cultures, indicating that the amounts of extracellular RTX toxin-like proteins are, in part, controlled by the abundance of the type I secretion apparatus. This is, to our knowledge, the first example of a two-component system mediating secretion of cytotoxin family proteins by controlling expression of the type I secretion proteins.

The circadian rhythmicity of hormone secretion, body temperature, and sleep/wakefulness results from an endogenous rhythm of neural activity generated by clock genes in the suprachiasmatic nucleus (SCN). One of these genes, Clock, has been considered essential for the generation of cellular rhythmicity centrally and in the periphery; however, melatonin-proficient [Clock.sup.[DELTA]19] + MEL mutant mice retain melatonin rhythmicity, suggesting that their central rhythmicity is intact. Here we show that melatonin production in these mutants was rhythmic in constant darkness and could be entrained by brief single daily light pulses. Under normal light-dark conditions, per2 and prokineticin2 (PK2) mRNA expression was rhythmic in the SCN of [Clock.sup.[DELTA]19] + MEL mice. Expression of Bma11 and npas2 was not altered, whereas per1 expression was arrhythmic. In contrast to the SCN, per1 and per2 expression, as well as Bmal1 expression in liver and skeletal muscle, together with plasma corticosterone, was arrhythmic in [Clock.sup.[DELTA]19] + MEL mutant mice in normal light-dark conditions. npas2 mRNA was also arrhythmic in liver but rhythmic in muscle. The Clock.sup.[DELTA]19] mutation does not abolish central rhythmicity and light entrainment, suggesting that a functional Clock homolog, possibly npas2, exists in the SCN. Nevertheless, the SCN of Clock.sup.[DELTA]19] + MEL mutant mice cannot maintain liver and muscle rhythmicity through rhythmic outputs, including melatonin secretion, in the absence of functional Clock expression in the tissues. Therefore, liver and muscle, but not SCN, have an absolute requirement for CLOCK, with as yet unknown Clock-independent factors able to generate the latter. circadian; melatonin; clock genes

Noninvasive imaging has been developed to measure gastric volumes. The relationship between gastric emptying and volume postprandially is unclear. The aims were to 1) develop a 3-dimensional (3D) single photon emission-computed tomography (SPECT) method to simultaneously measure gastric volume and emptying postprandially, 2) describe the course of gastric volume change during emptying of the meal, and 3) assess a 3D method measuring gastric emptying. In 30 healthy volunteers, we used [sup.111]In-planar and [sup.99m]Tc-SPECT imaging to estimate gastric emptying and volume after a radiolabeled meal. A customized analysis program of SPECT imaging assessed gastric emptying. A Bland-Altman plot assessed the performance of the new SPECT analysis compared with planar analysis. Gastric volume postprandially exceeds the fasting volume plus meal volume. The course of volume change and gastric emptying differ over time. Higher differences in volumes exist relative to fasting plus residual meal volumes at 15 min (median 763 vs. 568 ml, respectively, P < 0.001), 1 h (median 632 vs. 524 ml, P < 0.001), and 2 h (median 518 vs. 428 ml, P < 0.02), in contrast to similar volumes at 3 h (median 320 vs. 314 ml, P = 0.85). Analysis of SPECT imaging accurately measures gastric emptying compared with planar imaging with median differences of 1% (IQR -2.25 to 2.0) at 1 h, 1% (-3.25 to 2.25) at 2 h, and -2.5% (-4 to 0) at 3 h. Gastric volume exceeds meal volume during the first 2 postprandial hours, and simultaneous measurements of gastric volume and emptying can be achieved with a novel 3D SPECT method. stomach; accommodation; single photon emission computed tomography; imaging; gamma camera; three dimensional; secretion

AexT is an extracellular ADP ribosyltransferase produced by the fish pathogen Aeromonas salmonicida subsp. salmonicida. The protein is secreted by the bacterium via a recently identified type III secretion system. In this study, we have identified a further 12 open reading frames that possess high homology to genes encoding both structural and regulatory components of the Yersinia type III secretion apparatus. Using marker replacement mutagenesis of aopB, the A. salmonicida subsp, salmonicida homologue of yopB in Yersinia, we demonstrate that the bacterium translocates the AexT toxin directly into the cytosol of cultured fish cells via this type III secretion pathway. An acrV mutant of A. salmonicida subsp. salmonicida displays a calcium-blind phenotype, expressing and secreting significant amounts of AexT even in the presence of CaC[l.sub.2] concentrations as high as 10 mM. This acrV mutant is also unable to translocate AexT into the cytosol of fish cells, indicating AcrV is involved in the translocation process. Inactivation of either the aopB or acrV gene in A. salmonicida subsp. salmonicida (resulting in an inability to translocate AexT) is accompanied by a loss of cytotoxicity that can be restored by trans complementation. Finally, we present data indicating that preincubation of the wild-type bacteria with antibodies directed against recombinant AcrV-His protein provides fish cells protection against the toxic effects of the bacterium.

The virulence of Listeria monocytogenes is directly related to its ability to spread from cell to cell without leaving the intracellular milieu. During cell-to-cell spread, bacteria become temporarily confined to secondary vacuoles. Among the bacterial factors involved in escape from these vacuoles is a secreted broad-range phospholipase C (PC-PLC), the activation of which requires processing of an N-terminal prodomain. Mpl, a secreted metalloprotease of Listeria, is involved in the proteolytic activation of PC-PLC. We previously showed that, during intracellular growth, bacteria maintain a pool of PC-PLC that is not accessible to antibodies and that is rapidly released in its active form in response to a decrease in pH. pH-regulated release of active PC-PLC is Mpl dependent. To further characterize the mechanism regulating secretion of PC-PLC, the bacterial localization of PC-PLC and Mpl was investigated. Both proteins were detected in the bacterial supernatant and lysate with no apparent changes in molecular weight. Extraction of bacteria-associated PC-PLC and Mpl required cell wall hydrolysis, but there was no indication that either protein was covalently bound to the bacterial cell wall. Results from pulse-chase experiments performed with infected macrophages indicated that the rate of synthesis of PC-PLC exceeded the rate of translocation across the bacterial cell wall and confirmed that the pool of PC-PLC associated with bacteria was efficiently activated and secreted upon acidification of the host cell cytosol. These data suggest that bacterially associated PC-PLC and Mpl localize at the cell wall-membrane interface and that translocation of PC-PLC across the bacterial cell wall is rate limiting, resulting in the formation of a bacterially associated pool of PC-PLC that would readily be accessible for activation and release into nascent secondary vacuoles.

LamB-LacZ fusion proteins have classically been used in studies of the general secretion pathway of Escherichia coli. Here we describe how increasing signal sequence hydrophobicity routes LamB-LacZ Hyb42-1 to the signal recognition particle (SRP) pathway. Secretion of this hydrophobic fusion variant (H*LamB-LacZ) was reduced in the absence of fully functional Ffh and Ffs, and the translocator jamming caused by Hyb42-1 was prevented by efficient delivery of the fusion to the periplasm. Finally, we found that in the absence of the ribosome-associated chaperone, trigger factor (Tig), LamB-LacZ localized to the periplasm in a SecA-dependent, SRP-independent fashion. Collectively, our results provide compelling in vivo evidence that there is an SRP-dependent cotranslational targeting mechanism in E. coli and argue against a role for trigger factor in pathway discrimination.

The type III secretion system (TTSS) encoded by Salmonella typhimurium pathogenicity island 2(SPI-2) is expressed after bacterial entry into host cells. The SPI-2 TTSS secretes the translocon components SseBCD, which translocate across the vacuolar membrane a number of effector proteins whose action is required for intracellular bacterial replication. Several of these effectors, including SifA and SifB, are encoded outside SPI-2. The two-component regulatory system SsrA--SsrB, encoded within SPI-2, controls the expression of components of the SPI-2 TTSS apparatus as well as its translocated effectors. The expression of SsrA--B is in turn regulated by the OmpR--EnvZ two-component system, by direct binding of OmpR to the ssrAB promoter. Several environmental signals have been shown to induce in vitro expression of genes regulated by the SsrA--B or OmpR--EnvZ systems. In this work, immunoblotting and flow cytometry were used to analyse the roles of SsrA--B and OmpR--EnvZ in coupling different environmental signals to changes in expression of a SPI-2 TTSS translocon component (SseB) and two effector genes (sifA and sifB). Using single and double mutant strains the relative contribution of each regulatory system to the response generated by low osmolarity, acidic pH or the absence of [Ca.sup.2=] was determined. SsrA--B was found to be essential for the induction of SPI-2 gene expression in response to each of these individual signals. OmpR--EnvZ was found to play a minor role in sensing these signals and to require a functional SsrA--B system to mediate their effect on SPI-2 TTSS gene expression.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen implicated in a variety of devastating conditions. Its flexibility as a pathogen is attributed to a myriad of virulence factors and regulatory elements that respond to prevailing environmental conditions. ExoS and ExoT are type III secreted effector proteins, regulated by the transcriptional activator ExsA, that can inhibit invasion of epithelial cells by cytotoxic strains of P. aeruginosa. This study sought to understand why invasive strains, which can secrete both ExoS and ExoT, still invade epithelial cells. The results showed that LasA and elastase (LasB), which are regulated by the Las and Rhl quorum-sensing systems, modulated P. aeruginosa invasion. Mutation of lasA and/or lasB reduced P. aeruginosa invasion, which was not fully restored by extracellularly added LasB, P. aeruginosa conditioned medium containing LasA and LasB, or EGTA pretreatment of cells. This indicated that protease effects on invasion involved factors additional to tight junction disruption and subsequent alterations to cell polarity. Upon mutation of lasA and/or lasB, steady-state levels of ExoS and ExoT were increased in culture medium of P. aeruginosa grown under conditions stimulatory for these toxins. The increase in ExoS was significantly correlated with reduced invasion. In vitro experiments showed that purified LasB degraded recombinant ExoS. Taken together, these studies suggest a mechanism by which invasive strains can synthesize inhibitors of invasion, ExoS and ExoT, yet still invade epithelial cells. By this mechanism, LasA and LasB decrease the levels of the toxins directly or indirectly, and thus reduce inhibition of invasion.

The authors have previously reported the identification of novel signal peptides (SPs) from Lactococcus lactis using transposon insertion. Of these, SP310 caused the highest level of secretion. However, the levels were lower than those obtained using the signal peptide from Usp45 (SPUSP), the major secreted lactococcal protein. In this study, site-directed mutagenesis of signal peptide SP310 was used to investigate the effect of amino acid alterations on lactococcal secretion and to improve secretion efficiency. Several mutated SPs caused higher secretion. This increase in secretion was due to modifications in the cleavage region. In fermenter experiments, the signal peptide SP310mut2 resulted in an extracellular Staphylococcus aureus nuclease (Nuc) yield which was 45% higher than that with the natural SP310. Surprisingly, increasing the hydrophobicity of the hydrophobic core or increasing the number of positively charged amino acids in the N-terminal region of SP310 decreased secretion. High extracellular yields of Nuc resulted from more efficient secretion, as strains with less efficient SPs accumulated more intracellular SP-Nuc precursor.

Many Gram-negative pathogens employ a specific secretion pathway, termed type III secretion, to deliver virulence effector proteins directly to the membranes and cytosol of host eukaryotic cells. Subsequent functions of many effector proteins delivered in this manner result in subversion of host-signalling pathways to facilitate bacterial entry, survival and dissemination to neighbouring cells and tissues. Whereas the secreted components of type III secretion system (TTSSs) from different pathogens are structurally and functionally diverse, the structural components and the secretion apparatus itself are largely conserved. TTSSs are large macromolecular assemblies built through interactions between protein components of hundreds of individual subunits. The goal of this project was to screen, using the standard yeast two-hybrid system, pair-wise interactions between components of the enteropathogenic Escherichia coliTTSS. To this end 37 of the 41 genes encoded by the LEE pathogenicity island were cloned into both yeast two-hybrid system vectors and all possible permutations of interacting protein pairs were screened for. This paper reports the identification of 22 novel interactions, including interactions between innermembrane structural TTSS proteins; between the type III secreted translocator protein EspD and structural TTSS proteins; between established and putative chaperones and their cognate secreted proteins; and between proteins of undefined function.

The synaptotagmins (syts) are a family of membrane proteins proposed to regulate membrane traffic in neuronal and nonneuronal cells. In neurons, the [Ca.sup.2+]-sensing ability of syt I is critical for fusion of docked synaptic vesicles with the plasma membrane in response to stimulation. Several putative [Ca.sup.2+]-syt effectors have been identified, but in most cases the functional significance of these interactions remains unknown. Here, we have used recombinant C2 domains derived from the cytoplasmic domains of syts I-XI to interfere with endogenous syt-effector interactions during [Ca.sup.2+]-triggered exocytosis from cracked PC12 cells. Inhibition was closely correlated with syntaxin-SNAP-25 and phosphatidylinositol 4,5-bisphosphate (PI[P.sub.2])--binding activity. Moreover, we measured the expression levels of endogenous syts in PC12 cells; the major isoforms are I and IX, with trace levels of VII. As expected, if syts I and IX function as [Ca.sup.2+] sensors, fragments from these isoforms blocked secretion. These data suggest that syts trigger fusion via their [Ca.sup.2+]-regulated interactions with t-SNAREs and PI[P.sub.2], target molecules known to play critical roles in exocytosis.

The objective of the present study was to evaluate the secretion of testosterone (T) in bulls in response to the administration of varying doses of bovine LH (bLH) during the four seasons of the year. Five adult bulls (4 yr of age) were treated with an amount of bLH that was estimated to induce a 5 ng/mL amplitude pulse of LH in blood serum on five consecutive days around the spring equinox, summer solstice, fall equinox, and winter solstice. Five hours after this dose, bulls were treated with bLH in amounts that were estimated to induce a 0.25, 0.5, 1, 2, or 4 ng/mL amplitude LH pulse in blood serum in a Latin square design. Blood samples were collected for 5 h after administration of a dose of bLH that was estimated to induce the 5-ng amplitude LH pulse, and for 3 h after administration of the variable doses of bLH, and were then assayed for concentrations of T. Average concentrations and amplitude of T release after doses of bLH that were estimated to induce the 5-ng amplitude LH pulses were greater during the spring and summer than during the winter (P < 0.05). The area under the release curve (AUC) was greater during the spring than during the winter (P < 0.05). During the 3 h after treatment with the variable doses of bLH, T response was affected by dose (P < 0.001) and season (P < 0.001), but there was no dose x season interaction. Testosterone response increased in a dose-dependent fashion for all variables studied. The greatest average concentrations of T and AUC were observed in the spring compared with the fall and winter (P < 0.05). These data support our working hypothesis that testes of bulls are more responsive in releasing T in response to bLH stimulation in the spring and summer compared with the winter; however, there were no changes in sensitivity of the testes to LH during different seasons of the year as indicated by the lack of a dose of bLH x season interaction. Key Words: Cattle, Male Animals, Seasonal Variation, Testes, Testosterone

Genetic alterations in tumor cells often lead to the emergence of growth-stimulatory autocrine and paracrine signals, involving over-expression of secreted peptide growth factors, cytokines, and hormones. Increased levels of these soluble proteins may be exploited for cancer diagnosis and management or as points of therapeutic intervention. Here, we combined the use of controlled vocabulary terms and sequence-based algorithms to predict genes encoding secreted proteins from among [approximately equal to] 12,500 sequences represented on oligonucleotide microarrays. Expression of these genes was queried in 150 carcinomas from 10 anatomic sites of origin and compared with 46 normal tissues derived from the corresponding sites of tumor origin and other body tissues and organs. Of 74 different genes identified as overexpressed in cancer tissues, several encode proteins with demonstrated clinical diagnostic application, such as [alpha]-fetoprotein in liver carcinoma, and kallikreins 6 and 10 in ovarian cancer, or therapeutic utility, such as gastrin-releasing peptide/bombesin in lung carcinomas. We show that several of the other candidate genes encode proteins with high levels of tumor-associated expression by immunohistochemistry on tissue microarrays and further demonstrate significantly elevated levels of another novel candidate protein, macrophage inhibitory cytokine 1, a distant member of the tranforming growth factor-[beta] superfamily, in the serum of patients with metastatic prostate, breast, and colorectal carcinomas. Our results suggest that the combination of annotation/protein sequence analysis, transcript profiling, immunohistochemistry, and immunoassay is a powerful approach for delineating candidate biomarkers with potential clinical significance and may be broadly applicable to other human diseases. gene expression | microarray | genome ontology | sequence analysis | immunohistochemistry

In yeast, growth and organelle segregation requires formin-dependent assembly of polarized actin cables. These tracks are used by myosin Vs to deliver secretory vesicles for cell growth, organelles for their segregation, and mRNA for fate determination. Several specific receptors have been identified that interact with the cargo-binding tails of the myosin Vs. A recent study implicates specific degradation in the bud of the vacuolar receptor, Vac17, as a mechanism for cell cycle-regulated segregation of this organelle.

During the development of the Drosophila wing, the activity of the Notch signalling pathway is required to establish and maintain the organizing activity at the dorsoventral boundary (D/V boundary). At early stages, the activity of the pathway is restricted to a small stripe straddling the D/V boundary, and the establishment of this activity domain requires the secreted molecule fringe (fng). The activity domain will be established symmetrically at each side of the boundary of Fng-expressing and non-expressing cells. Here, I present evidence that the Drosophila tumour-suppressor gene lethal (2) gaint discs (lgd) is required to restrict the activity of Notch to the D/V boundary. In the absence of lgd function, the activity of Notch expands from its initial domain at the D/V boundary. This expansion requires the presence of at least one of the Notch ligands, which can activate Notch more efficiently in the mutants. The results further suggest that Lgd appears to act as a general repressor of Notch activity, because it also affects vein, eye, and bristle development. Keywords: Tumour suppressor genes; l(2)giant discs; Wing development; Notch-signalling; Serrate; Abruptex; Delta

Classical cell biology teaches that exocytosis causes the membrane of exocytic vesicles to disperse into the cell surface and that a cell must later retrieve by molecular sorting whatever membrane components it wishes to keep inside. We have tested whether this view applies to secretory granules in intact PC-12 cells. Three granule proteins were labeled with fluorescent proteins in different colors, and two-color evanescent-field microscopy was used to view single granules during and after exocytosis. Whereas neuropeptide Y was lost from granules in seconds, tissue plasminogen activator (tPA) and the membrane protein phogrin remained at the granule site for over 1 min, thus providing markers for postexocytic granules. When tPA was imaged simultaneously with cyan fluorescent protein (CFP) as a cytosolic marker, the volume occupied by the granule appeared as a dark spot where it excluded CFP. The spot remained even after tPA reported exocytosis, indicating that granules failed to flatten into the cell surface. Phogrin was labeled with GFP at its luminal end and used to sense the pH in granules. When exocytosis caused the acidic granule interior to neutralize, GFP-phogrin at first brightened and later dimmed again as the interior separated from the extracellular space and reacidified. Reacidification and dimming could be reversed by application of [NH.sub.4]CI. We conclude that most granules reseal in PC-12 cells | evanescent-field microscopy | endocytosis | kiss and run

Potassium secretory flux ([J.sub.K]) by the distal nephron is regulated by systemic and luminal factors. In the present investigation, [J.sub.K] was measured with a double-barreled [K.sup.+] electrode during paired microperfusion of superficial segments of the rat distal nephron. We used control solutions (100 mM NaCl, pH 7.0) and experimental solutions in which [Cl.sup.-] had been replaced with a less permeant anion and/or pH had been increased to 8.0. [J.sub.K] increased when [Cl.sup.-] was replaced by either acetate (~37%), sulfate (~32%), or bicarbonate (~62%), and also when the pH of the control perfusate was increased (~26%). The majority (80%) of acetate-stimulated [J.sub.K] was [Ba.sup.2+] sensitive, but furosemide (1 mM) further reduced secretion (~10% of total), suggesting that [K.sup.+]-[Cl.sup.-] cotransport was operative. Progressive reduction in luminal [Cl.sup.-] concentration from 100 to 20 to 2 mM caused increments in [J.sub.K] that were abolished by inhibitors of [K.sup.+]-[Cl.sup.-] cortransport, i.e., furosemide and [(dihydroindenyl)oxy]alkanoic acid. Increasing the pH of the luminal perfusion fluid also increased [J.sub.K] even in the presence of [Ba.sup.2+], suggesting that this effect cannot be accounted for only by [K.sup.+] channel modulation of [K.sup.+] secretion in the distal nephron of the rat. Collectively, these data suggest a role for [K.sup.+]-[Cl.sup.-] cotransport in distal nephron [K.sup.+] secretion. distal tubule; collecting duct; potassium secretion; postassium-chloride cotransport

Research has been conducted on gamma-secretase whose substrate indlude Notch and amyloid precursor protein. Results demonstrate that both Notch and amyloid precursor protein undergo cleavages within the transmembrane domain.

Research has been conducted on secreted glycoprotein thrombospondin-1. The anti-angiogenic activity of this glycoprotein which is localized to the thrombospondin type 1 repeats/domains has been investigated, and the highly conserved essential features of these thrombospondin type 1 repeats/domains have been characterized and discussed.

Dietary fish oil has immunomodulatory effects that are mediated in part by its effects on cytokines. Secretion of the inflammatory and the anti-inflammatory cytokines tumor necrosis factor (TNF) and interleukin (IL)-10 by murine resident peritoneal macrophages was monitored after ex vive stimulation with lipopolysaccharide. Macrophages were obtained from mice fed a corn oil diet containing 200 g/kg corn oil or a fish oil diet containing 180 g/kg fish oil and 20 g/kg corn oil. Dietary fish oil increased secretion of the proinflammatory cytokine, TNF, but decreased secretion of the anti-inflammatory cytokine, IL-10. The cytokines appeared in the medium after 1.5 h and peaked at 6-12 h. Neutralizing antibodies against TNF and IL-10 had little effect on secretion of the other cytokine, indicating that the effects of fish oil on TNF and IL-10 secretion by these cells are independent of one another. Furthermore, although inhibiting prostaglandin production enhanced TNF secretion by macrophages from mice fed the corn oil diet, it did not affect IL-10 secretion by macrophages in this group. Blocking leukotriene [B.sub.4] production also did not affect IL-10 secretion in macrophages from mice fed a nonpurified diet. These results demonstrate that fish oil has an overall pro-inflammatory effect given its effects on secretion of both inflammatory and anti-inflammatory cytokines by resident peritoneal macrophages. J. Nutr. 132: 3740-3743, 2002. KEY WORDS: * fish oil * cytokines * tumor necrosis factor * interleukin 10 * mice

Gastric secretion of hydrochloric acid requires protons and chloride, yet the mechanisms and regulation of gastric chloride secretion remain unclear. We developed an in vivo technique to simultaneously measure acid/base and chloride secretion into the gastric lumen of anesthetized rats. The cannulated stomach lumen was perfused with weakly pH-buffered chloride-free solution containing a chloride-sensitive fluorophore [5 [micro]M N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE)]. Gastric acid and chloride secretion was detected in gastric effluents by 1) flow-through pH electrode and 2) MQAE fluorescence. Gastric effluent was also collected at 1-min intervals for independent determination of chloride amount by chloridometer. In all conditions, both optical and chemical determinations of chloride report similar amounts of secreted chloride. During luminal perfusion with pH 5 solution, net acid and chloride secretion into the lumen was observed. Pentagastrin stimulated both secretions. In contrast, proton pump inhibition (omeprazole) caused alkalinization of the gastric effluent, but chloride secretion was not diminished. During luminal pH 3 perfusion, net alkali secretion was observed, and chloride secretion at luminal pH 3 was greater than pH 5. When tissue is pretreated with omeprazole at luminal pH 3, the addition of prostaglandin E2 synchronously stimulates both alkali and chloride secretion. Results suggest that both acid and alkali secretions are separately coupled with chloride secretion. proton pump, fluorescence, rat, stomach gastric luminal pH

Research has been conducted on Saccharomyces cerevisiae polyploid cells. The expression of glucoamylase in S. cerevisiae cells has been carried out, and the results demonstrate that glucoamylase genes are integrated into delta-sequence.

Research has been conducted on Helicobacter pylori secreted proteins. The identification of the proteins released by the mechanisms other than lysis has been carried out via the in vitro analysis, and the results indicate that after being treated with nalidixic acid some proteins were still enriched greater than 10-fold in the medium suggesting that they could be released by secretion.

Research has been conducted on fish pathogen Aeromonas salmonicida subsp. salmonicida. The hypothesis that type III secretion system is present in this bacterium has been tested, and the results demonstrate that knockout mutagenesis of the ycsV homologue in A.salmonicida subsp. salmonicida prevents AexT toxin secretion.

Research has been conducted on Yersinia enterocolitica yop gene expression, negatively regulated by yscM1 and yscM2. Results demonstrate that yopD and lcrH are required for yscM1 and yscM2 function and that these four genes act synergistically at the same regulatory step.

Biodirected epitaxial nanodeposition of polymers was achieved on a template with an oriented molecular surface. Acetobacter xylinum synthesized a ribbon of cellulose I microfibrils onto a fixed, nematic ordered substrate of glucan chains with unique surface characteristics. The substrate directed the orientation of the motion due to the inverse force of the secretion during biosynthesis, and the microfibrils were aligned along the orientation of the molecular template. Using real-time video analysis, the patterns and rates of deposition were elucidated. Field emission scanning electron microscopy revealed that a strong molecular interaction allowed for the deposition of nascent biosynthesized 3.5-nm cellulose microfibrils with inter-microfibrillar spacings of 7-8 nm on the surface of the template. The cellulose was deposited parallel to the molecular orientation of the template. Directed cellulose synthesis and ordered movement of cells were observed only by using a nematic ordered substrate made from cellulose, and not from ordered crystalline cellulose substrates or ordered cellulose-related synthetic polymers such as polyvinyl alcohol. This unique relationship between directed biosynthesis and the ordered fabrication from the nano to the micro scales could lead to new methodologies for the design of functional materials with desired nanostructures.

Results indicate that class V comprising htrA and htrB genes are heat-inducible and are regulatd by the CssR-CssS two-component regulatory system in Bacillus subtilis. Data show that transcription of the cssRS operon is autoregulated and induced by secretion stress and expression of htrA, htrB, cssR, and cssS is responsive to secretion stress.

The two-hybrid protein screen, DivIVa and FtsZ, can be used to target green fluorescent proteins to cell division sites in both Escherichia coli and Agrobacteium tumefaciens. Data suggest that such cell-location specific two-hybrid screens are useful to study interaction among cytosolic and membrane proteins in bacteria.

Acute hypertension inhibits proximal tubule (PT) fluid reabsorption. The resultant increase in end proximal flow rate provides the error signal to mediate tubuloglomerular feedback autoregulation of renal blood flow and glomerular filtration rate and suppresses renal renin secretion. To test whether the suppression of the renin-angiotensin system during acute hypertension affects the magnitude of the inhibition of PT fluid and sodium reabsorption, plasma ANG II levels were clamped by infusion of the angiotensin-converting enzyme (ACE) inhibitor captopril (12 [micro]g/min) and ANG II after pretreatment with the bradykinin [B.sub.2] receptor blocker HOE-140 (100 [micro]g/kg bolus). Because ACE also degrades bradykinin, HOE-140 was included to block effect of accumulating vasodilatory bradykinins during captopril infusion. HOE-140 increased the sensitivity of arterial blood pressure to ANG II: after captopril infusion without HOE-140, 20 ng*[kg.sup.-1]*[min.sup.-1] ANG II had no pressor effect, whereas with HOE-140, 20 ng*[kg.sup.-1]*[min.sup.-1] ANG II increased blood pressure from 104 [+ or -] 4 to 140 [+ or -] 6 mmHg. ANG II infused at 2 ng*[kg.sup.-1]*[min.sup.-1] had no pressor effect after captopril and HOE-140 infusion ('ANG II clamp'). When blood pressure was acutely increased 50-60 mmHg by arterial constriction without ANG II clamp, urine output and endogenous lithium clearance increased 4.0- and 6.7-fold, respectively. With ANG II clamp, the effects of acute hypertension were reduced 50%: urine output and endogenous lithium clearance increased two- and threefold, respectively. We conclude that HOE-140, an inhibitor of the [B.sub.2] receptor, potentiates the sensitivity of arterial pressure to ANG II and that clamping systemic ANG II levels during acute hypertension blunts the magnitude of the pressure diuretic response. captopril; HOE-140; bradykinin receptor; endogenous lithium clearance

Regulated secretion in exocrine and neuroendocrine cells occurs through exocytosis of secretory granules and the subsequent release of stored small molecules and proteins. The introduction of biophysical techniques with high temporal and spatial resolution, and the identification of [Ca.sup.2+]-dependent and -independent 'docking' and 'fusion' proteins, has greatly enhanced our understanding of exocytosis. The cloning of families of ion channel proteins, including intracellular ion channels, has also revived interest in the role of secretory granule ion channels in exocytotic secretion. Thus secretory granules of pancreatic acinar cell express a ClC-2 [Cl.sup.-] channel, a HC[O.sup.-.sub.3]-permeable member of the CLCA [Ca.sup.2+]-dependent anion channel family, and a KCNQ1 [K.sup.+] channel. Evidence suggests that these channels may facilitate the release of digestive enzymes and/or prevent exocytosed granules from collapsing during 'kiss and run' recycling. In pancreatic [beta]-cells, a granular ClC-3 [Cl.sup.-] channel provides a shunt pathway for a vacuolar-type [H.sup.+]-ATPase. Acidification 'primes' the granules for [Ca.sup.2+]-dependent exocytosis and release of insulin. In summary, secretory granules are equipped with specific sets of ion channels, which modulate regulated exocytosis and the release of macromolecules. These channels could represent excellent targets for therapeutic interventions to control exocytotic secretion in relevant diseases, such as pancreatitis, cystic fibrosis, or diabetes mellitus. acini; [beta]-cells; secretion; zymogen granules; sulfonylureas

Numerous bacterial pathogens use type IV secretion systems (T4SS) to deliver virulence factors directly to the cytoplasm of plant, animal, and human host cells. Here, evidence for interactions among components of the Agrobacterium tumefaciens vir-encoded T4SS is presented. The results derive from a high-resolution yeast two-hybrid assay, in which a library of small peptide domains of T4SS components was screened for interactions. The use of small peptides overcomes problems associated with assaying for interactions involving membrane-associated proteins. We established interactions between VirB11 (an inner membrane pore-forming protein), VirB9 (a periplasmic protein), and VirB7 (an outer membrane-associated lipoprotein and putative pilus component). We provide evidence for an interaction pathway, among conserved members of a T4SS, spanning the A. tumefaciens envelope and including a potential pore protein. In addition, we have determined interactions between VirB1 (a lytic transglycosylase likely involved in the local remodeling of the peptidoglycan) and primarily VirB8, but also VirB4, VirB10, and VirB11 (proteins likely to assemble the core structure of the T4SS). VirB4 interacts with VirB8, VirB10, and VirB11, also establishing a connection to the core components. The identification of these interactions suggests a model for assembly of the T4SS.

Research has been conducted on the serpin endopin 2. Results indicate that this endopin cross-class inhibits cysteine and serine proteases and that its inhibitory functions can occur in the regulatory secretion pathway.

Amyloid [beta]-peptide (A[beta]) is generated by the consecutive cleavages of [beta]-and [gamma]-secretase. The intramembraneous [gamma]-secretase cleavage critically depends on the activity of presenilins (PS1 and PS2). Although there is evidence that PSs are aspartyl proteases with [gamma]-secretase activity, it remains controversial whether their subcellular localization overlaps with the cellular sites of A[beta] production. We now demonstrate that biologically active GFP-tagged PS1 as well as endogenous PS1 are targeted to the plasma membrane (PM) of living cells. On the way to the PM, PS1 binds to nicastrin (Nct), an essential component of the [gamma]-secretase complex. This complex is targeted through the secretory pathway where PS1-bound Nct becomes endoglycosidase H resistant. Moreover, surface-biotinylated Nct can be coimmunoprecipitated with PS1 antibodies, demonstrating that this complex is located to cellular sites with [gamma]-secretase activity. Inactivating PS1 or PS2 function by mutagenesis of one of the critical aspartate residues or by [gamma]-secretase inhibitors results in delayed reinternalization of the [beta]-amyloid precursor protein and its accumulation at the cell surface. Our data suggest that PS is targeted as a biologically active complex with Nct through the secretory pathway to the cell surface and suggest a dual function of PS in [gamma]-secretase processing and in trafficking.

Research has been conducted on Yersinia enterocolitica. The translation fusion of ubiquitin or dihydrofolate reductase yop genes produces hybrid proteins which cannot be secreted and the results indicate that the hybrids' folding prevents their transport but does not affect the type III secretion in other Yops.

Research has been conducted on the oligomeric forms of colicin A detected on sodium dodecyl sulfate gels with unheated samples from colicin A-producing Escherichia coli cells. The export mechanism and physiological role of colicin A heat-labile species are discussed.

Dictyostelium Crp is a member of the cyclin-dependent kinase (Cdk) family of proteins. It is most related in sequence to mammalian Cdk5, which unlike other members of the family, has functions that are unrelated to the cell cycle. In order to better understand the function of Crp in Dictyostelium, we overexpressed a dominant negative form, Crp-D144N, under the control of the actin 15 promoter. Cells overexpressing Crp-D144N exhibit a reduced growth rate in suspension culture and reduced rates of fluid-phase endocytosis and phagocytosis. There is no reduction in Cdc2 kinase activity in extracts from cells overexpressing Crp-D144N, suggesting that the growth defect is not due to inhibition of Cdc2. In addition to the growth defect, the act15::crp-D144N transformants aggregate at a slower rate than wild-type cells and form large aggregation streams. These eventually break up to form small aggregates and most of these do not produce mature fruiting bodies. The aggregation defect is fully reversed in the presence of wild-type cells but terminal differentiation is only partially rescued. In act15::crp-D144N transformants, the countin component of the counting factor, a secreted protein complex that regulates the breakup of streams, mostly appears outside the cell as degradation products and the reduced level of the intact protein may at least partially account for the initial formation of the large aggregation streams. Our observations indicate that Crp is important for both endocytosis and efflux and that defects in these functions lead to reduced growth and aberrant development. Key Words: Dictyostelium; Cdk5; Cdc2-related kinase; differentiation; growth.

Research has been conducted on Lactobacillus bulgaricus extracellular proteinase. The efficient expression of the full-length or truncated forms of this proteinase with different secretion patterns carried out via the use of the expression vector constructs is reported.

Research has been conducted on Saccharomyces cerevisiae K28 killer strains. The toxicity of K28 has been investigated via the site-directed mutagenesis of the pptox gene and the phenotypic effects on K28 immunity and toxin secretion have been examined and discussed.