Focus on centrin in normal and altered human spermatozoa.

This review provides details on the role of centrin in human spermatozoa and in various forms of male infertility. Centrin is a calcium (Ca2+)-binding phosphoprotein that is located in the centrioles - which are typical structures of the sperm connecting piece and play a key role in centrosome dynamics during sperm morphogenesis - as well as in zygotes and early embryos during spindle assembly. In humans, three different centrin genes encoding three isoforms have been discovered. Centrin 1, the only one expressed in spermatozoa, seems to be lost inside the oocyte after fertilization. The sperm connecting piece is characterized by the presence of numerous proteins including centrin, that deserves particular attention because, in humans, it is enriched during maturation of the centrioles. In normal sperm, centrin 1 is visible as two distinct spots in the head-tail junction; however, in some defective spermatozoa, centrin 1 distribution is altered. Centrin has been studied in humans and animal models. Its mutations may lead to several structural alterations, such as serious defects in the connective piece and, subsequently, fertilization failure or incomplete embryonic development. However, the effects of these abnormalities on male fertility have not been fully studied. Because the presence and the function of centrin in the sperm connecting piece appears important for reproductive success, additional studies are needed to bring medical benefits in resolving some cases of idiopathic infertility.

Sequence-Specific Gene Silencing of acrA in the Multi-drug Efflux System AcrAB Induces Sensitivity in Drug-Resistant Klebsiella pneumoniae.

Multi-drug efflux is one of the resistant determinants in Klebsiella pneumoniae that are encountered in a broad range of clinically relevant antimicrobial agents. An alternative method to strategically induce sensitivity in drug-resistant K. pneumoniae and improve the efficacy of the existing antibiotics is the need of the hour. Hence, an antisense RNA was designed against the acrA gene of the AcrAB-TolC efflux system in a drug-resistant isolate of K. pneumoniae obtained from a blood culture. Minimum inhibitory concentration by E test demonstrated that the antisense RNA could significantly increase the susceptibility of previously resistant K. pneumoniae toward ciprofloxacin (CIP) and co-trimoxazole. Real-time PCR determined the ability of the antisense RNA to inhibit the expression of the acrA-mRNA. The wild-type K. pneumoniae showed increased growth in the presence of CIP, while, under the same condition, the growth of the antisense RNA-treated K. pneumoniae was inhibited up till 12 h. In the presence of co-trimoxazole, delayed growth rate of the antisense RNA-treated K. pneumoniae was seen, in comparison to that of the wild-type K. pneumoniae and also a fourfold reduction was noted in the expression of the efflux gene acrA. Our results underscore the potential of the acrA antisense RNA as an alternative therapeutic against multi-drug-resistant K. pneumoniae.

Expression analysis of centrin gene in promastigote and amastigote forms of leishmania infantum iranian isolates: a promising target for live attenuated vaccine development against canine leishmaniasis.

BACKGROUND: Leishmania parasites express various essential proteins in different growth phases (logarithmic/stationary) and forms (promastigote/amastigote). Targeting the genes encoding such proteins paves the way for controlling these parasites. Centrin is an essential gene, which its protein product seems to be vital for the proliferation of Leishmania parasites. Herein, this study was contrived to analyze the expression level of the centrin gene in different growth phases and forms of Leishmania infantum (L. infantum) parasites isolated from various endemic areas of canine leishmaniasis (CanL) in Iran. RESULTS: All three collected isolates were identified as L. infantum using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Real-time reverse transcription (RT)-PCR revealed a statistically significant up-regulation (3.13-fold) in the logarithmic phase promastigotes compared to stationary ones (p < 0.01), whereas centrin was expressed equally in intracellular amastigotes at different time points during cell culture. Also, our finding revealed a slight up-regulation of the centrin gene (1.22-fold) in the intracellular amastigotes compared to logarithmic phase promastigotes, which was found statistically non-significant (p > 0.05). CONCLUSIONS: Centrin gene in Iranian isolates of L. infantum is more expressed in exponential than stationary phases and seems to be considered as a promising target in the development of a genetically modified live attenuated vaccine for CanL control.

A helical inner scaffold provides a structural basis for centriole cohesion.

The ninefold radial arrangement of microtubule triplets (MTTs) is the hallmark of the centriole, a conserved organelle crucial for the formation of centrosomes and cilia. Although strong cohesion between MTTs is critical to resist forces applied by ciliary beating and the mitotic spindle, how the centriole maintains its structural integrity is not known. Using cryo-electron tomography and subtomogram averaging of centrioles from four evolutionarily distant species, we found that MTTs are bound together by a helical inner scaffold covering ~70% of the centriole length that maintains MTTs cohesion under compressive forces. Ultrastructure Expansion Microscopy (U-ExM) indicated that POC5, POC1B, FAM161A, and Centrin-2 localize to the scaffold structure along the inner wall of the centriole MTTs. Moreover, we established that these four proteins interact with each other to form a complex that binds microtubules. Together, our results provide a structural and molecular basis for centriole cohesion and geometry.

The roles of Centrin 2 and Dynein Light Chain 8a in apical secretory organelles discharge of Toxoplasma gondii.

To efficiently enter host cells, apicomplexan parasites such as Toxoplasma gondii rely on an apical complex composed of tubulin-based structures as well as two sets of secretory organelles named micronemes and rhoptries. The trafficking and docking of these organelles to the apical pole of the parasite is crucial for the discharge of their contents. Here, we describe two proteins typically associated with microtubules, Centrin 2 (CEN2) and Dynein Light Chain 8a (DLC8a), that are required for efficient host cell invasion. CEN2 localizes to four different compartments, and remarkably, conditional depletion of the protein occurs in stepwise manner, sequentially depleting the protein pools from each location. This phenomenon allowed us to discern the essential function of the apical pool of CEN2 for microneme secretion, motility, invasion and egress. DLC8a localizes to the conoid, and its depletion also perturbs microneme exocytosis in addition to the apical docking of the rhoptry organelles, causing a severe defect in host cell invasion. Phenotypic characterization of CEN2 and DLC8a indicates that while both proteins participate in microneme secretion, they likely act at different steps along the cascade of events leading to organelle exocytosis.

Trypanosoma brucei centrin5 is enriched in the flagellum and interacts with other centrins in a calcium-dependent manner.

Centrin is an evolutionarily conserved EF-hand-containing protein, which is present in eukaryotic organisms as diverse as algae, yeast, and humans. Centrins are associated with the microtubule-organizing center and with centrosome-related structures, such as basal bodies in flagellar and ciliated cells, and the spindle pole body in yeast. Five centrin genes have been identified in Trypanosoma brucei (T. brucei), a protozoan parasite that causes sleeping sickness in humans and nagana in cattle in sub-Saharan Africa. In the present study, we identified that centrin5 of T. brucei (TbCentrin5) is localized throughout the cytosol and nucleus and enriched in the flagellum. We further identified that TbCentrin5 binds Ca2+ ions with a high affinity and constructed a model of TbCentrin5 bound by Ca2+ ions. Meanwhile, we observed that TbCentrin5 interacts with TbCentrin1, TbCentrin3, and TbCentrin4 and that the interactions are Ca2+ -dependent, suggesting that TbCentrin5 is able to form different complexes with other TbCentrins to participate in relevant cellular processes. Our study provides a foundation for better understanding of the biological roles of TbCentrin5.

Biology of multiciliated cells.

Multiciliated cells (MCCs) are specialized in fluid propulsion through directional beating of myriads of superficial motile cilia, which rest on modified centrioles named basal bodies. MCCs are found throughout metazoans, and serve functions as diverse as feeding and locomotion in marine organisms, as well as mucus clearance, cerebrospinal fluid circulation, and egg transportation in mammals. Impaired MCC differentiation or activity causes diseases characterized by severe chronic airway infections and reduced fertility. Through studies in Xenopus and mouse mainly, MCC biology has made significant progress on several fronts in recent years. The gene regulatory network that controls MCC specification and differentiation has been deciphered to a large extent. The enigmatic deuterosomes, which serve as centriole amplification platforms in vertebrate MCCs, have started to be studied at the molecular level. Principles of ciliary beating coordination within and between MCCs have been identified.

Human IFT52 mutations uncover a novel role for the protein in microtubule dynamics and centrosome cohesion.

Mutations in genes encoding components of the intraflagellar transport (IFT) complexes have previously been associated with a spectrum of diseases collectively termed ciliopathies. Ciliopathies relate to defects in the formation or function of the cilium, a sensory or motile organelle present on the surface of most cell types. IFT52 is a key component of the IFT-B complex and ensures the interaction of the two subcomplexes, IFT-B1 and IFT-B2. Here, we report novel IFT52 biallelic mutations in cases with a short-rib thoracic dysplasia (SRTD) or a congenital anomaly of kidney and urinary tract (CAKUT). Combining in vitro and in vivo studies in zebrafish, we showed that SRTD-associated missense mutation impairs IFT-B complex assembly and IFT-B2 ciliary localization, resulting in decreased cilia length. In comparison, CAKUT-associated missense mutation has a mild pathogenicity, thus explaining the lack of skeletal defects in CAKUT case. In parallel, we demonstrated that the previously reported homozygous nonsense IFT52 mutation associated with Sensenbrenner syndrome [Girisha et al. (2016) A homozygous nonsense variant in IFT52 is associated with a human skeletal ciliopathy. Clin. Genet., 90, 536-539] leads to exon skipping and results in a partially functional protein. Finally, our work uncovered a novel role for IFT52 in microtubule network regulation. We showed that IFT52 interacts and partially co-localized with centrin at the distal end of centrioles where it is involved in its recruitment and/or maintenance. Alteration of this function likely contributes to centriole splitting observed in Ift52-/- cells. Altogether, our findings allow a better comprehensive genotype-phenotype correlation among IFT52-related cases and revealed a novel, extra-ciliary role for IFT52, i.e. disruption may contribute to pathophysiological mechanisms.

BBS4 is required for intraflagellar transport coordination and basal body number in mammalian olfactory cilia.

Bardet-Beidl syndrome (BBS) manifests from genetic mutations encoding for one or more BBS proteins. BBS4 loss impacts olfactory ciliation and odor detection, yet the cellular mechanisms remain unclear. Here, we report that Bbs4-/- mice exhibit shorter and fewer olfactory sensory neuron (OSN) cilia despite retaining odorant receptor localization. Within Bbs4-/- OSN cilia, we observed asynchronous rates of IFT-A/B particle movements, indicating miscoordination in IFT complex trafficking. Within the OSN dendritic knob, the basal bodies are dynamic, with incorporation of ectopically expressed centrin-2 and γ-tubulin occurring after nascent ciliogenesis. Importantly, BBS4 loss results in the reduction of basal body numbers separate from cilia loss. Adenoviral expression of BBS4 restored OSN cilia lengths and was sufficient to re-establish odor detection, but failed to rescue ciliary and basal body numbers. Our results yield a model for the plurality of BBS4 functions in OSNs that includes intraciliary and periciliary roles that can explain the loss of cilia and penetrance of ciliopathy phenotypes in olfactory neurons.

Crystal structure of the trimeric N-terminal domain of ciliate Euplotes octocarinatus centrin binding with calcium ions.

Centrin is a member of the EF-hand superfamily of calcium-binding proteins, a highly conserved eukaryotic protein that binds to Ca2+ . Its self-assembly plays a causative role in the fiber contraction that is associated with the cell division cycle and ciliogenesis. In this study, the crystal structure of N-terminal domain of ciliate Euplotes octocarinatus centrin (N-EoCen) was determined by using the selenomethionine single-wavelength anomalous dispersion method. The protein molecules formed homotrimers. Every protomer had two putative Ca2+ ion-binding sites I and II, protomer A, and C bound one Ca2+ ion, while protomer B bound two Ca2+ ions. A novel binding site III was observed and the Ca2+ ion was located at the center of the homotrimer. Several hydrogen bonds, electrostatic, and hydrophobic interactions between the protomers contributed to the formation of the oligomer. Structural studies provided insight into the foundation for centrin aggregation and the roles of calcium ions.

Alterations of growth rate and gene expression levels of UPEC by antibiotics at sub-MIC.

The host is the main environment for bacteria, and they also expose to many antibiotics during the treatment of infectious diseases in host body. In this study, it was aimed to investigate possible changes in growth rate and expression levels of three virulence genes (foc/foc, cnf1, and usp) in a uropathogenic E. coli standard strain within the presence of ciprofloxacin, nitrofurantoin, and trimethoprim-sulfamethoxazole. The UPEC C7 strain was grown on tryptic soy broth-TSB (control), TSB + ciprofloxacin, TSB + nitrofurantoin, and TSB + trimethoprim-sulfamethoxazole for determination of both growth rate and gene expression level. Antibiotics were added according to their sub-minimal inhibition concentrations. E-test was used to determine MIC values of antibiotics. Growth changes were measured in absorbance 600 nm during 24-h period. Total RNA isolations were performed after incubation for 24 h at 37 °C. Gene expression levels were determined by quantitative PCR. Tukey's post hoc test was used for statistical analysis. According to absorbance values, it has been shown that only ciprofloxacin and trimethoprim-sulfamethoxazole have lead significant decrease on growth rate. We also detected statistically significant differences in each gene expression levels for all antibiotics via relative quantification analysis. Fold changes in gene expression was found 0.65, 1.42, 0.23 for foc/foc gene; 0.01, 0.01, 2.84 for cnf1 gene; and 0.1, 0.01, 0.01 for usp gene in the presence of ciprofloxacin, nitrofurantoin, and trimethoprim/sulfamethoxazole, respectively. This investigation has shown that antibiotics can play a role as an environmental factor which may determine the pathogenicity of bacteria in vivo.

Modulation of XPC peptide on binding Tb3+ to Euplotes octocarinatus centrin.

Centrins are Ca2+-binding proteins found throughout eukaryotic organisms. Xeroderma pigmentosum group C protein (XPC), a dominant component of the nuclear excision repair (NER) pathway, is a critical target protein of centrins. A 22-residue peptide (K842-R863) from XPC was used to investigate the effect of metal ions (Ca2+ and Tb3+) on the peptide binding of Euplotes octocarinatus centrin (EoCen) by isothermal titration calorimetry (ITC) and fluorescence spectroscopy. ITC and tryptophan spectrofluorimetric titrations revealed that metal ions (Ca2+ and Tb3+) could enhance the affinity between EoCen and the XPC peptide, and the enhanced effects were closely related to the ion potential of metal ions. Since the ion potential of Tb3+ (e/r = 0.0325) is larger than that of Ca2+ (e/r = 0.0202), the conformational change in the protein induced by Tb3+ is larger than that induced by Ca2+, and the enhanced affinity of Tb3+ is stronger than that of Ca2+. This interaction was driven by enthalpy in the presence of EDTA and enthalpy and entropy in the presence of Ca2+ or Tb3+. Similar to that observed in the presence of EDTA, the N-terminal domain did not participate in the interaction with the XPC peptide even in the presence of metal ions. Resonance light scattering (RLS) and the band shift in native polyacrylamide gel electrophoresis (PAGE) suggested that peptide binding resulted in the dissociation of EoCen aggregates and complex formation via the monomer-peptide form. Tb3+-Sensitized emission suggested that peptide binding in turn also had an impact on the Tb3+ binding of the protein: the C-terminal domain was slightly strengthened and the N-terminal domain was weakened about 225 fold. RLS and native PAGE indicated that the self-assembly induced by Tb3+ binding to the N-terminal domain of EoCen was inhibited in the presence of the XPC peptide. This study elucidates the molecular mechanism of EoCen function in the cellular context.

Mechanisms of Resistance to Folate Pathway Inhibitors in Burkholderia pseudomallei: Deviation from the Norm.

The trimethoprim and sulfamethoxazole combination, co-trimoxazole, plays a vital role in the treatment of Burkholderia pseudomallei infections. Previous studies demonstrated that the B. pseudomallei BpeEF-OprC efflux pump confers widespread trimethoprim resistance in clinical and environmental isolates, but this is not accompanied by significant resistance to co-trimoxazole. Using the excluded select-agent strain B. pseudomallei Bp82, we now show that in vitro acquired trimethoprim versus co-trimoxazole resistance is mainly mediated by constitutive BpeEF-OprC expression due to bpeT mutations or by BpeEF-OprC overexpression due to bpeS mutations. Mutations in bpeT affect the carboxy-terminal effector-binding domain of the BpeT LysR-type activator protein. Trimethoprim resistance can also be mediated by dihydrofolate reductase (FolA) target mutations, but this occurs rarely unless BpeEF-OprC is absent. BpeS is a transcriptional regulator that is 62% identical to BpeT. Mutations affecting the BpeS DNA-binding or carboxy-terminal effector-binding domains result in constitutive BpeEF-OprC overexpression, leading to trimethoprim and sulfamethoxazole efflux and thus to co-trimoxazole resistance. The majority of laboratory-selected co-trimoxazole-resistant mutants often also contain mutations in folM, encoding a pterin reductase. Genetic analyses of these mutants established that both bpeS mutations and folM mutations contribute to co-trimoxazole resistance, although the exact role of folM remains to be determined. Mutations affecting bpeT, bpeS, and folM are common in co-trimoxazole-resistant clinical isolates, indicating that mutations affecting these genes are clinically significant. Co-trimoxazole resistance in B. pseudomallei is a complex phenomenon, which may explain why resistance to this drug is rare in this bacterium.IMPORTANCEBurkholderia pseudomallei causes melioidosis, a tropical disease that is difficult to treat. The bacterium's resistance to antibiotics limits therapeutic options. The paucity of orally available drugs further complicates therapy. The oral drug of choice is co-trimoxazole, a combination of trimethoprim and sulfamethoxazole. These antibiotics target two distinct enzymes, FolA (dihydrofolate reductase) and FolP (dihydropteroate synthase), in the bacterial tetrahydrofolate biosynthetic pathway. Although co-trimoxazole resistance is minimized due to two-target inhibition, bacterial resistance due to folA and folP mutations does occur. Co-trimoxazole resistance in B. pseudomallei is rare and has not yet been studied. Co-trimoxazole resistance in this bacterium employs a novel strategy involving differential regulation of BpeEF-OprC efflux pump expression that determines the drug resistance profile. Contributing are mutations affecting folA, but not folP, and folM, a folate pathway-associated gene whose function is not yet well understood and which has not been previously implicated in folate inhibitor resistance in clinical isolates.

Identification and characterization of new members of the SXT/R391 family of integrative and conjugative elements (ICEs) in Proteus mirabilis.

Integrative and conjugative elements (ICEs) are self-transmissible chromosomal mobile elements that play significant roles in the dissemination of antimicrobial resistance genes. Identification of the structures and functions of ICEs, particularly those in pathogens, improves understanding of the dissemination of antimicrobial resistance. This study identified new members of the sulfamethoxazole-trimethoprim (SXT)/R391 family of ICEs that could confer multi-drug resistance in the opportunistic pathogen Proteus mirabilis, characterized their genetic structures, and explored their evolutionary connection with other members of this family of ICEs. Three new members of the SXT/R391 family of ICEs were detected in six of 77 P. mirabilis strains isolated in China: ICEPmiChn2 (one strain), ICEPmiChn3 (one strain) and ICEPmiChn4 (three strains). All three new ICEs harbour antimicrobial resistance genes from diverse origins, suggesting their capability in acquiring foreign genes and serving as important carriers for antimicrobial resistance genes. Structural analysis showed that ICEPmiChn3 is a particularly interesting and unique ICE that has lost core genes involved in conjugation, and could not transfer to other cells via conjugation. This finding confirmed the key roles of these missing genes in conjugation. Further phylogenetic analysis suggested that ICEs in geographically close strains are also connected evolutionarily, and ICEPmiChn3 lost its conjugation cassette from a former mobile ICE. The identification and characterization of the three new members of the SXT/R391 family of ICEs in this work leads to suggestions of core ICE genes essential for conjugation, and extends understanding on the structures of ICEs, evolutionary relationships between ICEs, and the antimicrobial resistance mechanisms of P. mirabilis.

Myosin-dependent cell-cell communication controls synchronicity of division in acute and chronic stages of Toxoplasma gondii.

The obligate intracellular parasite Toxoplasma gondii possesses a repertoire of 11 myosins. Three class XIV motors participate in motility, invasion and egress, whereas the class XXII myosin F is implicated in organelle positioning and inheritance of the apicoplast. Here we provide evidence that TgUNC acts as a chaperone dedicated to the folding, assembly and function of all Toxoplasma myosins. The conditional ablation of TgUNC recapitulates the phenome of the known myosins and uncovers two functions in parasite basal complex constriction and synchronized division within the parasitophorous vacuole. We identify myosin J and centrin 2 as essential for the constriction. We demonstrate the existence of an intravacuolar cell-cell communication ensuring synchronized division, a process dependent on myosin I. This connectivity contributes to the delayed death phenotype resulting from loss of the apicoplast. Cell-cell communication is lost in activated macrophages and during bradyzoite differentiation resulting in asynchronized, slow division in the cysts.

Prp40 Homolog A Is a Novel Centrin Target.

Pre-mRNA processing protein 40 (Prp40) is a nuclear protein that has a role in pre-mRNA splicing. Prp40 possesses two leucine-rich nuclear export signals, but little is known about the function of Prp40 in the export process. Another protein that has a role in protein export is centrin, a member of the EF-hand superfamily of Ca2+-binding proteins. Prp40 was found to be a centrin target by yeast-two-hybrid screening using both Homo sapiens centrin 2 (Hscen2) and Chlamydomonas reinhardtii centrin (Crcen). We identified a centrin-binding site within H. sapiens Prp40 homolog A (HsPrp40A), which contains a hydrophobic triad W1L4L8 that is known to be important in the interaction with centrin. This centrin-binding site is highly conserved within the first nuclear export signal consensus sequence identified in Saccharomyces cerevisiae Prp40. Here, we examine the interaction of HsPrp40A peptide (HsPrp40Ap) with both Hscen2 and Crcen by isothermal titration calorimetry. We employed the thermodynamic parameterization to estimate the polar and apolar surface area of the interface. In addition, we have defined the molecular mechanism of thermally induced unfolding and dissociation of the Crcen-HsPrp40Ap complex using two-dimensional infrared correlation spectroscopy. These complementary techniques showed for the first time, to our knowledge, that HsPrp40Ap interacts with centrin in vitro, supporting a coupled functional role for these proteins in pre-mRNA splicing.

Application of rapid in vitro co-culture system of macrophages and T-cell subsets to assess the immunogenicity of dogs vaccinated with live attenuated Leishmania donovani centrin deleted parasites (LdCen-/-).

BACKGROUND: Live attenuated Leishmania donovani parasites as LdCen(-/-) were shown to confer protective immunity against Leishmania infection in mice, hamsters, and dogs. Strong immunogenicity in dogs vaccinated with LdCen(-/-) has been previously reported, including increased antibody response favoring Th1 response lymphoproliferative responses, CD4(+) and CD8(+) T-cells activation, increased levels of Th1 and reduction of Th2 cytokines, in addition to a significant reduction in parasite burden after 18 and 24 months post virulent parasite challenge. METHODS: Aimed at validating a new method using in vitro co-culture systems with macrophages and purified CD4(+) or CD8(+) or CD4(+):CD8(+) T-cells of immunized dogs with both LdCen(-/-) and Leishmune® to assess microbicide capacity of macrophages and the immune response profile as the production of IFN-γ, TNF-α, IL-12, IL-4 and IL-10 cytokines. RESULTS AND DISCUSSION: Our data showed co-cultures of macrophages and purified T-cells from dogs immunized with LdCen(-/-) and challenged with L. infantum were able to identify high microbicidal activity, especially in the co-culture using CD4(+) T-cells, as compared to the Leishmune® group. Similarly, co-cultures with CD8(+) T-cells or CD4(+):CD8(+) T-cells in both experimental groups were able to detect a reduction in the parasite burden in L. infantum infected macrophages. Moreover, co-cultures using CD4(+) or CD8(+) or CD4(+):CD8(+) T-cells from immunized dogs with both LdCen(-/-) and Leishmune® were able to identify higher levels of IFN-γ and IL-12 cytokines, reduced levels of IL-4 and IL-10, and a higher IFN-γ/IL-10 ratio. While the highest IFN-γ levels and IFN-γ/IL-10 ratio were the hallmarks of LdCen(-/-) group in the co-culture using CD4(+) T-cells, resulting in strong reduction of parasitism, the Leishmune® immunization presented a differential production of TNF-α in the co-culture using CD4(+):CD8(+) T-cells. CONCLUSION: The distinct conditions of co-culture systems were validated and able to detect the induction of immune protection. The method described in this study applied a new, more accurate approach and was able to yield laboratory parameters useful to test and monitor the immunogenicity and efficacy of Leishmania vaccines in dogs.

Duplication of the Yeast Spindle Pole Body Once per Cell Cycle.

The yeast spindle pole body (SPB) is the functional equivalent of the mammalian centrosome. Centrosomes and SPBs duplicate exactly once per cell cycle by mechanisms that use the mother structure as a platform for the assembly of the daughter. The conserved Sfi1 and centrin proteins are essential components of the SPB duplication process. Sfi1 is an elongated molecule that has, in its center, 20 to 23 binding sites for the Ca(2+)-binding protein centrin. In the yeastSaccharomyces cerevisiae, all Sfi1 N termini are in contact with the mother SPB whereas the free C termini are distal to it. During S phase and early mitosis, cyclin-dependent kinase 1 (Cdk1) phosphorylation of mainly serine residues in the Sfi1 C termini blocks the initiation of SPB duplication ("off" state). Upon anaphase onset, the phosphatase Cdc14 dephosphorylates Sfi1 ("on" state) to promote antiparallel and shifted incorporation of cytoplasmic Sfi1 molecules into the half-bridge layer, which thereby elongates into the bridge. The Sfi1 C termini of the two Sfi1 layers localize in the bridge center, whereas the N termini of the newly assembled Sfi1 molecules are distal to the mother SPB. These free Sfi1 N termini then assemble the new SPB in G1phase. Recruitment of Sfi1 molecules into the anaphase SPB and bridge formation were also observed inSchizosaccharomyces pombe, suggesting that the Sfi1 bridge cycle is conserved between the two organisms. Thus, restricting SPB duplication to one event per cell cycle requires only an oscillation between Cdk1 kinase and Cdc14 phosphatase activities. This clockwork regulates the "on"/"off" state of the Sfi1-centrin receiver.

Why sulfonamides are contraindicated in Rocky Mountain spotted fever.

Sulfonamide antibiotics are not effective for the treatment of Rocky Mountain spotted fever (RMSF). Patients suspected of having RMSF based on history and physical exam should be treated with doxycycline and not a sulfonamide to avoid increased morbidity and mortality.