Paternity analysis reveals wide pollen dispersal and high multiple paternity in a small isolated population of the bird-pollinated Eucalyptus caesia (Myrtaceae).

Optimal foraging behaviour by nectavores is expected to result in a leptokurtic pollen dispersal distribution and predominantly near-neighbour mating. However, complex social interactions among nectarivorous birds may result in different mating patterns to those typically observed in insect-pollinated plants. Mating system, realised pollen dispersal and spatial genetic structure were examined in the bird-pollinated Eucalyptus caesia, a species characterised by small, geographically disjunct populations. Nine microsatellite markers were used to genotype an entire adult stand and 181 seeds from 28 capsules collected from 6 trees. Mating system analysis using MLTR revealed moderate to high outcrossing (tm=0.479-0.806) and low estimates of correlated paternity (rp=0.136±s.e. 0.048). Paternity analysis revealed high outcrossing rates (mean=0.72) and high multiple paternity, with 64 different sires identified for 181 seeds. There was a significant negative relationship between the frequency of outcross mating and distance between mating pairs. Realised mating events were more frequent than expected with random mating for plants <40 m apart. The overall distribution of pollen dispersal distances was platykurtic. Despite extensive pollen dispersal within the stand, three genetic clusters were detected by STRUCTURE analysis. These genetic clusters were strongly differentiated yet geographically interspersed, hypothesised to be a consequence of rare recruitment events coupled with extreme longevity. We suggest that extensive polyandry and pollen dispersal is a consequence of pollination by highly mobile honeyeaters and may buffer E. caesia against the loss of genetic diversity predicted for small and genetically isolated populations.

The digestive morphophysiology of wild, free-living, giraffes.

We have measured rumen-complex (rumen, reticulum, omasum, abomasum) and intestine (small and large combined) mass in 32 wild giraffes of both sexes with body masses ranging from 289 to 1441 kg, and parotid gland mass, tongue length and mass, masseter and mandible mass in 9 other giraffes ranging in body mass from 181 to 1396 kg. We have estimated metabolic and energy production rates, feed intake and home range size. Interspecific analysis of mature ruminants show that components of the digestive system increase linearly (Mb(1)) or positively allometric (Mb(>1)) with body mass while variables associated with feed intake scale with metabolic rate (Mb(.75)). Conversely, in giraffes ontogenetic increases in rumen-complex mass were negatively allometric (Mb(<1)), and increases in intestine mass, parotid gland mass, masseter mass, and mandible mass were isometric (Mb(1)). The relative masseter muscle mass (0.14% of Mb) and the relative parotid mass (0.03% of Mb) are smaller than in other ruminants. Increases in tongue length scale with head length(0.72) and Mb(.32) and tongue mass with Mb(.69). Absolute mass of the gastrointestinal tract increased throughout growth but its relative mass declined from 20% to 15% of Mb. Rumen-complex fermentation provides ca 43% of daily energy needs, large intestine fermentation 24% and 33% by digestion of soluble carbohydrates, proteins, and lipids. Dry matter intake (kg) was 2.4% of body mass in juveniles and 1.6% in adults. Energy requirements increased from 35 Mj/day to 190 Mj/day. Browse production rate sustains a core home range of 2.2-11.8 km(2).

Microsatellite primers for Australian recreationally and commercially important estuarine fishes.

Eight primer sets that amplify microsatellite DNA loci were developed for the estuarine fishes, dusky flathead Platycephalus fuscus and sand whiting Sillago ciliata, which are targets of recreational and commercial fishers on the east coast of Australia. Surveys across these loci revealed remarkably similar levels of genetic diversity within samples of c. 22 P. fuscus and S. ciliata from each of the two estuaries. These loci will be useful in determining the genetic stock structure of these two species.

Is the post-disturbance composition of a plant population determined by selection for outcrossed seedlings or by the composition of the seedbank?

Seedbanks are expected to buffer populations against disturbances, such as fire, that could alter the genetic composition of smaller, ephemeral adult populations. However, seedling genotypes may be influenced by the spatially heterogeneous nature of both the seedbank and the disturbance (for example, germination may vary with local disturbance) and also by selection acting on germination and post-germination performance. We used microsatellite-DNA surveys of seedlings emerging from the soil-stored seedbanks of Grevillea macleayana after wildfire to compare diversity and spatial structure in seedlings and adults, and through resampling of the seedling data set, to determine whether the resultant adult population reflected the effects of selection or random seedling mortality. The large post-fire seedling cohorts captured the full allelic diversity of the pre-fire adult population. However, we found a mismatch in the genotypic structure of adults and seedlings. Seedlings displayed larger heterozygous deficits than adults; however, over the ensuing 11 years, seedling heterozygosity eventually matched values for the pre-fire adults. Increasing heterozygosity among adults has generally been attributed to heterosis and/or reduction in Wahlund effects via self-thinning. Resampling of early post-fire seedlings to generate samples of equivalent size to survivors at 11 years showed that increases in heterozygosity must be driven by selection favouring outcrossed seed. This finding is important in an evolutionary context but also has implications for the restoration of natural or managed populations where a seedbank is a viable source of recruits.

Gamete compatibility between marine and estuarine Acanthopagrus spp. (Sparidae) and their hybrids.

On Australia's south-east coast, hybridization between estuary-restricted black bream Acanthopagrus butcheri Munro and its migratory coastal congener yellowfin bream Acanthopagrus australis (Günther) has led to estuarine populations largely composed of hybrids that are most genetically similar to A. butcheri. The fertilization success achieved when ova of estuary-caught A. butcheri were fertilized with the cryogenically preserved sperm of either ocean-caught A. australis or estuary-caught A. butcheri-like was compared. The experimental crosses, which by chance included both pure parental and hybrid bream, revealed no evidence that gametic incompatibility provides a barrier to fertilization among both pure species and their hybrids.

Evolutionary impacts of hybridization and interspecific gene flow on an obligately estuarine fish.

For free-spawning estuarine taxa, gene flow among estuaries may occur via hybridization with mobile congeners. This phenomenon has rarely been investigated, but is probably susceptible to anthropogenic disturbance. In eastern Australia, the estuarine Black Bream Acanthopagrus butcheri and marine Yellowfin Bream Acanthopagrus australis have overlapping distributions and the potential to hybridize. We used surveys of microsatellite and mtDNA variation in 565 adults from 25 estuaries spanning their distributional range to characterize the species and their putative hybrids. Hybrids were widespread (68% of estuaries) and hybrid frequencies varied greatly among estuaries (0-58%). Most (88%) were classed as advanced generation backcrosses with A. butcheri and displayed A. butcheri mtDNA haplotypes. We found most hybrids in the three estuaries within the zone of sympatry (57%). Our study highlights the underemphasized importance of estuaries as sites of hybridization and suggests that hybridization is driven both by opportunity for contact and human activity.

Characterization of the EYE2 gene required for eyespot assembly in Chlamydomonas reinhardtii.

The unicellular biflagellate green alga Chlamydomonas reinhardtii can perceive light and respond by altering its swimming behavior. The eyespot is a specialized structure for sensing light, which is assembled de novo at every cell division from components located in two different cellular compartments. Photoreceptors and associated signal transduction components are localized in a discrete patch of the plasma membrane. This patch is tightly packed against an underlying sandwich of chloroplast membranes and carotenoid-filled lipid granules, which aids the cell in distinguishing light direction. In a prior screen for mutant strains with eyespot defects, the EYE2 locus was defined by the single eye2-1 allele. The mutant strain has no eyespot by light microscopy and has no organized carotenoid granule layers as judged by electron microscopy. Here we demonstrate that the eye2-1 mutant is capable of responding to light, although the strain is far less sensitive than wild type to low light intensities and orients imprecisely. Therefore, pigment granule layer assembly in the chloroplast is not required for photoreceptor localization in the plasma membrane. A plasmid-insertion mutagenesis screen yielded the eye2-2 allele, which allowed the isolation and characterization of the EYE2 gene. The EYE2 protein is a member of the thioredoxin superfamily. Site-directed mutagenesis of the active site cysteines demonstrated that EYE2 function in eyespot assembly is redox independent, similar to the auxiliary functions of other thioredoxin family members in protein folding and complex assembly.

Solid-phase synthesis of benzisothiazolones as serine protease inhibitors.

An efficient solid-phase synthesis of benzisothiazolone-1,1-dioxide-based serine protease inhibitors involving alkylation of carboxylic acids with N-(bromomethyl)benzisothiazolone-1,1-dioxide has been developed. An example using this procedure for preparation of a library of human mast cell tryptase inhibitors is described.

1,2-Benzisothiazol-3-one 1,1-dioxide inhibitors of human mast cell tryptase.

A library of compounds were prepared by reacting 2-(bromomethyl)-1, 2-benzisothiazol-3(2H)-one 1,1-dioxide (5) with commercially available carboxylic acids in the presence of potassium carbonate or a tertiary amine base. From this library, (1,1-dioxido-3-oxo-1, 2-benzisothiazol-2(3H)-yl)methyl N-[(phenylmethoxy)carbonyl]-beta-alanate (7b) emerged as a potent inhibitor of human mast cell tryptase (IC50 = 0.85 microM). Extension of the side chain of 7b by two carbons gave (1, 1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)methyl 5-[[(phenylmethoxy)carbonyl]amino]pentanoate (7d) which was an 8-fold more potent inhibitor (IC50 = 0.1 microM). Further modification of this series produced benzoic acid derivative (1, 1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)methyl 4-[[(phenylmethoxy)carbonyl]amino]benzoate (7n) which is the most potent inhibitor identified in this series (IC50 = 0.064 microM). These compounds exhibit time-dependent inhibition consistent with mechanism-based inhibition. For 7b, the initial enzyme velocity is not a saturable function of the inhibitor concentration and the initial Ki could not be determined (Ki > 10 microM). The steady-state rate constant, Ki, was determined to be 396 nM. On the other hand, compounds 7d and 7n are time-dependent inhibitors with a saturable initial complex. From these studies, an initial rate constant, Ki, for 7d and 7n was found to be 345 and 465 nM, respectively. The steady-state inhibition constants, Ki, for 7d and 7n were calculated to be 60 and 52 nM, respectively. Compound 7n is a 13-fold more potent inhibitor than 7b, and these kinetic studies indicate that the increase in inhibitory activity is due to an increase in initial affinity toward the enzyme and not an increase in chemical reactivity. These inhibitors generally show high selectivity for tryptase, being 40-fold weaker inhibitors of elastase, being 100-fold weaker against trypsin, and showing no inhibition against thrombin. These compounds are not inhibitors of thrombin, plasmin t-PA, urokinase, and factor Xa (IC50 > 33 microM). In the delayed-type hypersensitivity (DTH) mouse model, a model of skin inflammation, a 5% solution of 7d reduced edema by 69% compared to control animals.

Severe burns resulting from an exploding teat on a bottle of infant formula milk heated in a microwave oven.

Accidental injury to infants can result from the use of home microwave ovens. The spectrum of injury includes scald burns of the trachea, palate oropharynx and oesophagus due to aspiration and ingestion of foods that have been overheated. There is one previous case report of a child with second-degree burns of 6 per cent of his body area due to explosion of the plastic liner and nipple on a feeding bottle top. We present a case report of an infant who suffered 7 per cent full-thickness burns following explosion of the bottle teat and subsequent splattering with hot milk. He required mid-palmar amputation of his left hand including thumb, index and middle fingers. The full-thickness burns of left cheek and left shoulder were grafted but will result in significant scarring. It is common practice in many homes to heat infant feeds in the microwaves. We suggest that health professionals dealing with children need to be aware of the potential hazards so that appropriate education of parents can take place.

Crystallographic determination of the structures of human alpha-thrombin complexed with BMS-186282 and BMS-189090.

The crystallographic structures of the ternary complexes of human alpha-thrombin with hirugen (a sulfated hirudin fragment) and the small-molecule active site thrombin inhibitors BMS-186282 and BMS-189090 have been determined at 2.6 and 2.8 A. In both cases, the inhibitors, which adopt very similar bound conformations, bind in an antiparallel beta-strand arrangement relative to the thrombin main chain in a manner like that reported for PPACK, D-Phe-Pro-Arg-CH2Cl. They do, however, exhibit differences in the binding of the alkyl guanidine moiety in the specificity pocket. Numerous hydrophilic and hydrophobic interactions serve to stabilize the inhibitors in the binding pocket. Although PPACK forms covalent bonds to both serine and the histidine of the catalytic triad of thrombin, neither BMS-186282 nor BMS-189090 bind covalently and only BMS-186282 forms a hydrogen bond to the serine of the catalytic triad. Both inhibitors bind with high affinity (Ki = 79 nM and 3.6 nM, respectively) and are highly selective for thrombin over trypsin and other serine proteases.

Resolution of middle ear effusion in newborns.

OBJECTIVES: To determine the natural history of middle ear effusion (MEE) in newborns and compare the results of pneumatic otoscopy with tympanometry and acoustic reflex measurements in the evaluation of the middle ear of neonates. DESIGN: A descriptive natural history study with comparison of three evaluation methods for MEE. SETTING: County hospital nursery and pediatric clinic. PARTICIPANTS: Sixty-eight full-term, healthy neonates were studied on day 1 of life; 65 on day 2; and 24 on day 3. Thirty-eight infants returned at 2 weeks of age, and from this group, 23 returned at 2 months of age. INTERVENTIONS: Two independent observers performed otoscopy. An audiologist performed tympanometry and ipsilateral acoustic reflex measurements. Infants were evaluated daily from birth by all three methods for up to 3 days. MAIN OUTCOME MEASURE: The time to resolution of MEE as determined by three methods. RESULTS: In the first 3 hours of life, all babies examined had MEE diagnosed in both ears. By the third day, MEE apparently had resolved in 73% of ears by otoscopy, 88% by acoustic reflex measurements, and 92% by tympanometry. At 2 weeks, MEE was present by otoscopy in 13% (10/75 of ears). These were primarily newly acquired MEEs. Interobserver agreement by otoscopy as determined by kappa scores was moderate on days 1 and 3, poor on day 2, and excellent at 2 weeks and 2 months. CONCLUSIONS: Middle ear effusion diagnosed by otoscopy apparently resolves in 72 hours in most neonates. Interobserver agreement of otoscopists was excellent after babies were discharged from the nursery, suggesting that pneumatic otoscopy can be used to diagnose MEE in neonates this age. Most MEEs that are diagnosed 2 weeks and 2 months after birth are new and asymptomatic.

Structure of a retro-binding peptide inhibitor complexed with human alpha-thrombin.

The crystallographic structure of the ternary complex between human alpha-thrombin, hirugen and the peptidyl inhibitor Phe-alloThr-Phe-O-CH3, which is acylated at its N terminus with 4-guanidino butanoic acid (BMS-183507), has been determined at 2.6 A resolution. The structure reveals a unique "retro-binding" mode for this tripeptide active site inhibitor. The inhibitor binds with its alkyl-guanidine moiety in the primary specificity pocket and its two phenyl rings occupying the hydrophobic proximal and distal pockets of the thrombin active site. In this arrangement the backbone of the tripeptide forms a parallel beta-strand to the thrombin main-chain at the binding site. This is opposite to the orientation of the natural substrate, fibrinogen, and all the small active site-directed thrombin inhibitors whose bound structures have been previously reported. BMS-183507 is the first synthetic inhibitor proved to bind in a retro-binding fashion to thrombin, in a fashion similar to that of the N-terminal residues of the natural inhibitor hirudin. Furthermore, this new potent thrombin inhibitor (Ki = 17.2 nM) is selective for thrombin over other serine proteases tested and may be a template to be considered in designing hirudin-based thrombin inhibitors with interactions at the specificity pocket.

Cefixime compared with amoxicillin for treatment of acute otitis media.

Cefixime was compared with amoxicillin for treatment of acute otitis media in a randomized trial. Results of tympanocentesis on day 3 to 5 of therapy were used as the major outcome. Total daily doses were 8 mg/kg of cefixime and 40 mg/kg of amoxicillin. One hundred twenty-six patients were randomly assigned to receive treatment; 64 cultures grew pathogens. Pathogens were eradicated from the middle ear after 3 to 5 days of therapy in 27 (79.4%) of 34 children given amoxicillin and 26 (86.7%) of 30 children given cefixime (p = 0.47). When Streptococcus pneumoniae cases were analyzed, bacteriologic cure occurred in 14 (93.3%) of 15 children given amoxicillin and 12 (75%) of 16 given cefixime (p = 0.333). When cases of Haemophilus influenzae infection were analyzed, significantly more cures occurred with cefixime (10/10, 100%) than amoxicillin (8/13, 62%) (p = 0.046). Pathogens associated with failure of amoxicillin therapy were H. influenzae (five cases, two beta-lactamase-positive), S. pneumoniae (one case), and Moraxella catarrhalis (one case, beta-lactamase-positive). The four failures with cefixime therapy were all in patients infected with S. pneumoniae. Rates of rash, diarrhea, and vomiting were the same in both groups and did not necessitate stopping therapy. We conclude the following: (1) Cefixime and amoxicillin were equivalent in overall clinical and bacteriologic efficacy for otitis media. (2) Cefixime was more efficacious than amoxicillin in treating H. influenzae otitis media and should be preferred when H. influenzae is the suspected etiologic agent. (3) Side effects of both drugs were mild and equivalent.

Gamma globulin prophylaxis to reduce post-transfusion non-A, non-B hepatitis after cardiac surgery with cardiopulmonary bypass.

A prospective, randomized study of immune serum globulin (ISG) for prevention of post-transfusion hepatitis was performed on 196 patients (100 controls without gammaglobulin or placebo and 96 who received ISG) undergoing valve replacement or coronary artery bypass with extracorporeal circulation. The dose of ISG was 2 ml i.m. at premedication and 2 ml i.m. on postoperative day 3. Probable non-A, non-B hepatitis developed postoperatively in ten of the 100 controls and two of the 96 in the ISG group. Two ISG patients and three controls with non-A, non-B hepatitis still have increased serum aminotransferase values after 3-5 years, but liver biopsy revealed hepatitis, which histologically was very mild, in only two control and two ISG patients. Low-dose gamma globulin thus reduced the incidence of acute, probable non-A, non-B hepatitis in cardiac surgery with cardiopulmonary bypass.

Long-term follow-up of operative treatment for pulmonary metastases.

From 1954 onwards, 132 patients underwent 165 resections for pulmonary metastases. No other forms of therapy were adopted. The operative mortality was 7.6% (10 patients). After a minimum follow-up of 5 years, the overall survival rate was 20%. The 10- and 15-year survival rates were 6% and 3%, respectively. The major cause of late death was recurrence or spread of the primary disease in 102 patients (83.6%) while 5 patients died of reasons not related to cancer. Fifteen patients (12.3%) are still alive, 13 of whom have no clinical cancer. The presence of symptoms, the disease-free interval of less than 1 year and the number of metastases showed a statistically significant detrimental influence on survival (P less than 0.001, P less than 0.05 and P less than 0.046, respectively). The best 5-year survivals of 42%, 24%, 23% and 23% were noted for metastases from malignancies in the body of the uterus, kidney, bone and colon, respectively. Longterm survival or clinical cure can be achieved with surgery alone by an aggressive approach in selected malignancies.