Structure determination of the head-tail connector of bacteriophage phi29.

The head-tail connector of bacteriophage phi29 is composed of 12 36 kDa subunits with 12-fold symmetry. It is the central component of a rotary motor that packages the genomic dsDNA into preformed proheads. This motor consists of the head-tail connector, surrounded by a phi29-encoded, 174-base, RNA and a viral ATPase protein, both of which have fivefold symmetry in three-dimensional cryo-electron microscopy reconstructions. DNA is translocated into the prohead through a 36 A diameter pore in the center of the connector, where the DNA takes the role of a motor spindle. The helical nature of the DNA allows the rotational action of the connector to be transformed into a linear translation of the DNA. The crystal structure determination of connector crystals in space group C2 was initiated by molecular replacement, using an approximately 20 A resolution model derived from cryo-electron microscopy. The model phases were extended to 3.5 A resolution using 12-fold non-crystallographic symmetry averaging and solvent flattening. Although this electron density was not interpretable, the phases were adequate to locate the position of 24 mercury sites of a thimerosal heavy-atom derivative. The resultant 3.2 A single isomorphous replacement phases were improved using density modification, producing an interpretable electron-density map. The crystallographically refined structure was used as a molecular-replacement model to solve the structures of two other crystal forms of the connector molecule. One of these was in the same space group and almost isomorphous, whereas the other was in space group P2(1)2(1)2. The structural differences between the oligomeric connector molecules in the three crystal forms and between different monomers within each crystal show that the structure is relatively flexible, particularly in the protruding domain at the wide end of the connector. This domain probably acts as a bearing, allowing the connector to rotate within the pentagonal portal of the prohead during DNA packaging.

Comparison between antigenemia and a quantitative-competitive polymerase chain reaction for the diagnosis of cytomegalovirus infection after heart transplantation.

BACKGROUND: Antigenemia and quantitative polymerase chain reaction (PCR) are widely used for cytomegalovirus (CMV) diagnosis after heart transplantation due to their enhanced predictive values for disease detection when specific cut-off values are used. The purpose of this study was to compare, in the same patient setting, the predictive values of quantitative PCR and antigenemia for CMV disease detection, using specific cut-off values. METHODS: Thirty heart transplant receptors were ch prospectively monitored for active CMV infection and disease detection, using quantitative PCR and anti- po genemia. Positive and negative predictive values for pr CMV disease detection were calculated using cut-off pr values for both antigenemia (5 and 10 positive cells/300,000 neutrophils) and quantitative-PCR (50,000 and 100,000 copies/10(6) leukocytes). RESULTS: Active CMV infection was diagnosed in 93.3% of patients and CMV disease in 23.3%. The positive and negative predictive (%) values for CMV disease detection were 35/100 and 46.7/100, respectively, for quantitative PCR and antigenemia. Using 5 and 10 positive cells/300,000 neutrophils as cut-off values for antigenemia, the positive and negative predictive values (%) for disease detection were respectively 63.6/100 and 70/100. For quantitative PCR, the positive and th negative predictive values (%) for cut-off values of to 50,000 and 100,000 copies/10(6) leukocytes were 53.8/100 and 60/94.1, respectively. CONCLUSION: In our series, antigenemia and quantitative-PCR had enhanced and similar predictive values for CMV disease detection when specific cut-off values were used. The choice between these two methods for disease detection may rely less on their efficiency and more on the experience and familiarity with them.

Structure of the bacteriophage phi29 DNA packaging motor.

Motors generating mechanical force, powered by the hydrolysis of ATP, translocate double-stranded DNA into preformed capsids (proheads) of bacterial viruses and certain animal viruses. Here we describe the motor that packages the double-stranded DNA of the Bacillus subtilis bacteriophage phi29 into a precursor capsid. We determined the structure of the head-tail connector--the central component of the phi29 DNA packaging motor--to 3.2 A resolution by means of X-ray crystallography. We then fitted the connector into the electron densities of the prohead and of the partially packaged prohead as determined using cryo-electron microscopy and image reconstruction analysis. Our results suggest that the prohead plus dodecameric connector, prohead RNA, viral ATPase and DNA comprise a rotary motor with the head-prohead RNA-ATPase complex acting as a stator, the DNA acting as a spindle, and the connector as a ball-race. The helical nature of the DNA converts the rotary action of the connector into translation of the DNA.

Host range and variability of calcium binding by surface loops in the capsids of canine and feline parvoviruses.

Canine parvovirus (CPV) emerged in 1978 as a host range variant of feline panleukopenia virus (FPV). This change of host was mediated by the mutation of five residues on the surface of the capsid. CPV and FPV enter cells by endocytosis and can be taken up by many non-permissive cell lines, showing that their host range and tissue specificity are largely determined by events occurring after cell entry. We have determined the structures of a variety of strains of CPV and FPV at various pH values and in the presence or absence of Ca(2+). The largest structural difference was found to occur in a flexible surface loop, consisting of residues 359 to 375 of the capsid protein. This loop binds a divalent calcium ion in FPV and is adjacent to a double Ca(2+)-binding site, both in CPV and FPV. Residues within the loop and those associated with the double Ca(2+)-binding site were found to be essential for virus infectivity. The residues involved in the double Ca(2+)-binding site are conserved only in FPV and CPV. Our results show that the loop conformation and the associated Ca(2+)-binding are influenced by the Ca(2+) concentration, as well as pH. These changes are correlated with the ability of the virus to hemagglutinate erythrocytes. The co-localization of hemagglutinating activity and host range determinants on the virus surface implies that these properties may be functionally linked. We speculate that the flexible loop and surrounding regions are involved in binding an as yet unidentified host molecule and that this interaction influences host range.

The structure of an insect parvovirus (Galleria mellonella densovirus) at 3.7 A resolution.

BACKGROUND: Parvoviruses infect vertebrates, insects and crustaceans. Many arthropod parvoviruses (densoviruses) are highly pathogenic and kill approximately 90% of the host larvae within days, making them potentially effective as selective pesticides. Improved understanding of densoviral structure and function is therefore desirable. There are four different initiation sites for translation of the densovirus capsid protein mRNA, giving rise to the viral proteins VP1 to VP4. Sixty copies of the common, C-terminal domain make up the ordered part of the icosahedral capsid. RESULTS: The Galleria mellonella densovirus (GMDNV) capsid protein consists of a core beta-barrel motif, similar to that found in many other viral capsid proteins. The structure most closely resembles that of the vertebrate parvoviruses, but it has diverged beyond recognition in many of the long loop regions that constitute the surface features and intersubunit contacts. The N termini of twofold-related subunits have swapped their positions relative to those of the vertebrate parvoviruses. Unlike in the vertebrate parvoviruses, in GmDNV there is no continuous electron density in the channels running along the fivefold axes of the virus. Electron density corresponding to some of the single-stranded DNA genome is visible in the crystal structure, but it is not as well defined as in the vertebrate parvoviruses. CONCLUSIONS: The sequence of the glycine-rich motif, which occupies each of the channels along the fivefold axes in vertebrate viruses, is conserved between mammalian and insect parvoviruses. This motif may serve to externalize the N-terminal region of the single VP1 subunit per particle. The domain swapping of the N termini between insect and vertebrate parvoviruses may have the effect of increasing capsid stability in GmDNV.

Profiles of selected hormones during menstrual cycles of teenage athletes.

Concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone (P), 17 alpha-hydroxyprogesterone (17 alpha OHP), 17 beta-estradiol (E2), and prolactin (HPr) were monitored for one complete menstrual cycle in teenage swimmers, a gynecologically age-matched control group, and a group of fertile adult women. The swimmers experienced anovulatory menstrual cycles. The time from the LH surge to the onset of menses ("luteal" phase) was very short in the swimmers (4.5 +/- 0.6 days) in comparison with the lengths of these phases in the adults (13.4 +/- 1.7 days; P less than 0.05) and in the control group (7.8 +/- 3.0 days; P less than 0.05). In the follicular phase the swimmers' LH concentration was elevated and their FSH concentration was depressed in comparison with each of the other groups (P less than 0.05). Luteal phase FHS, P, E2, and 17 alpha OHP were also lower in the swimmers (P less than 0.05), as was HPr (0.05 greater than P less than 0.10). Gonadotropin concentrations and luteal phase P concentrations were not different (P greater than 0.05) in the adults and the control group. The present findings indicate that the corpora lutea in the swimmers were not functioning properly. It is likely that the swimmers' strenuous daily 2-4 h training regimen is implicated.

Modulation of pituitary responsiveness to LHRH by androgens in ovariectomized female rats.

The effect of androgens on pituitary response to luteinizing-hormore-releasing hormone (LHRH) and their ability to modify effects of 17beta-estradiol (E2) on pituitary responsiveness to LHRH were tested in ovariectomized rats maintained on a daily dose of 0.25 microgram estradiol benzoate per rat for 6 d before androgen administration. Testosterone propionate (TP) (4, 40, 400, or 4000 microgram per rat), administered 24 h before LHRH (500 ng per rat), had no significant effect on luteinizing hormone (LH) or follicle-stimulating hormone (FSH) response. Similar doses of dihydrotestosterone (DHT) did not significantly alter the LH response but significantly suppressed the FSH response. Even the lowest dose completely blocked the FSH response to LHRH. TP in combination with 4 or 400 microgram of E2 suppressed the stimulatory effect of E2 on both LH and FSH response to LHRH in a dose-related manner. DHT and E2 in combination affected LH response inconsistently, whereas their ratio determined FSH response; there was pronounced inhibition of FSH response in rats given high doses of DHT combined with low doses of E2; DHT inhibition of FSH response in animals receiving 4 microgram of DHT with 400 microgram E2 was partially overcome by the stimulatory effect of E2. Our results indicate that TP and DHT affect LH and FSH response to LHRH differently. The ratio of androgen to estrogen is important in determining the response to LHRH.

Prevention and suppression of post-partum lactation with 2-bromo-alpha-ergocryptine (CB-154).

Most clinical trials to date have demonstrated the effectiveness of 2-bromo-alpha-ergocryptine in suppressing the onset of puerperal lactation and confirmed the postulative mechanism of action by suppression of serum prolactin concentrations during its administration. The present study involving 112 patients was carried out to demonstrate the dose response and the timing of the administration. The drug was administered in two dose schedules of 1 mg t.i.d. and 2.5 mg b.i.d. commencing immediately after delivery and continued for 14 days. A third group of patients had 2.5 mg administered b.i.d. only after acute engorgement became a problem in the post-partum period. The drug was just as effective in the lower dose schedule and almost complete relief of pain and engorgement spared both the patient and the nursing staff many complaints. A dramatic relief was also obtained within 48 h even after engorgement had occurred and an overall incidence of rebound filling of 19% is similar to that reported by others.