Mannose-6-Phosphate Reductase, a Key Enzyme in Photoassimilate Partitioning, Is Abundant and Located in the Cytosol of Photosynthetically Active Cells of Celery (Apium graveolens L.) Source Leaves.

Mannitol, a major photosynthetic product and transport carbohydrate in many plants, accounts for approximately 50% of the carbon fixed by celery (Apium graveolens L.) leaves. Previous subfractionation studies of celery leaves indicated that the enzymes for mannitol synthesis were located in the cytosol, but these data are inconsistent with that published for the sites of sugar alcohol synthesis in other families and taxa, including apple (Malus) and a brown alga (Fucus). Using antibodies to a key synthetic enzyme, NADPH-dependent mannose-6-phosphate reductase (M6PR), and immunocytochemical techniques, we have resolved both the inter-cellular and intracellular sites of mannitol synthesis. In leaves, M6PR was found only in cells containing ribulose-1,5-bisphosphate carboxylase/oxygenase. M6PR was almost exclusively cytosolic in these cells, with the nucleus being the only organelle to show labeling. The key step in transport carbohydrate biosynthesis that is catalyzed by M6PR displays no apparent preferential association with vascular tissues or with the bundle sheath. These results show that M6PR and, thus, mannitol synthesis are closely associated with the distribution of photosynthetic carbon metabolism in celery leaves. The principal role of M6PR is, therefore, in the assimilation of carbon being exported from the chloroplast, and it seems unlikely that this enzyme plays even an indirect role in phloem loading of mannitol.

Gas Exchange and Carbon Partitioning in the Leaves of Celery (Apium graveolens L.) at Various Levels of Root Zone Salinity.

Both mannitol and sucrose (Suc) are primary photosynthetic products in celery (Apium graveolens L.). In other biological systems mannitol has been shown to serve as a compatible solute or osmoprotectant involved in stress tolerance. Although mannitol, like Suc, is translocated and serves as a reserve carbohydrate in celery, its role in stress tolerance has yet to be resolved. Mature celery plants exposed to low (25 mM NaCl), intermediate (100 mM NaCl), and high (300 mM NaCl) salinities displayed substantial salt tolerance. Shoot fresh weight was increased at low NaCl concentrations when compared with controls, and growth continued, although at slower rates, even after prolonged exposure to high salinities. Gas-exchange analyses showed that low NaCl levels had little or no effect on photosynthetic carbon assimilation (A), but at intermediate levels decreases in stomatal conductance limited A, and at the highest NaCl levels carboxylation capacity (as measured by analyses of the CO2 assimilation response to changing internal CO2 partial pressures) and electron transport (as indicated by fluorescence measurements) were the apparent prevailing limits to A. Increasing salinities up to 300 mM, however, increased mannitol accumulation and decreased Suc and starch pools in leaf tissues, e.g. the ratio of mannitol to Suc increased almost 10-fold. These changes were due in part to shifts in photosynthetic carbon partitioning (as measured by 14C labeling) from Suc into mannitol. Salt treatments increased the activity of mannose-6-phosphate reductase (M6PR), a key enzyme in mannitol biosynthesis, 6-fold in young leaves and 2-fold in fully expanded, mature leaves, but increases in M6PR protein were not apparent in the older leaves. Mannitol biosynthetic capacity (as measured by labeling rates) was maintained despite salt treatment, and relative partitioning into mannitol consequently increased despite decreased photosynthetic capacity. The results support a suggested role for mannitol accumulation in adaptation to and tolerance of salinity stress.

Production of polyclonal antibodies in rabbits is simplified using perforated plastic golf balls.

Production of polyclonal antibodies in the lumen of a perforated golf ball implanted surgically under the skin of a rabbit offers advantages over conventional techniques. Less stress is placed on the rabbit because bleeding is eliminated, complete adjuvants are not used and animal handling is minimized. The technique also offers the advantage that large amounts of antibody-containing fluid can be removed easily from the ball. In this report we describe the surgical protocol and demonstrate use of this technique to produce high-titered antibodies to plant and plant viral proteins.

Mongoose rabies in the Caribbean.

Mongooses (Herpestes auropunctatus) have been introduced into most of the larger Caribbean islands, some notable exceptions being Dominica, Tobago, and Montserrat. Rabies in Caribbean mongooses is present in Puerto Rico, Cuba, the Dominican Republic (and presumably Haiti), and Grenada. Bat rabies is known on Cuba, Grenada, and Trinidad, although mongooses found on Trinidad are free of the disease. None of the other islands is known to have rabies, although it could be present in sequestered bat populations. All reported case numbers of mongoose rabies in the Caribbean are underestimates, and available information is at best incomplete and at times fragmentary. Nevertheless, data are presented from the four affected islands. Mongoose reduction campaigns have been undertaken on Cuba and Grenada. In Cuba strychnine sulfate inoculated into labeled eggs is used, whereas in Grenada sodium fluoroacetate (1080) has been used in boiled cowhide baits. Mongoose poisoning is unsatisfactory and ineffective in the long-term. Because many mongooses naturally exposed to rabies virus develop serum neutralizing antibodies and are considered to be immunized, possibly for life, vaccination in the wild has been under consideration since the mid-1970s. Early attempts to produce a pill coated with ERA vaccine for enteric absorption in mongooses were not very successful, but new modified vaccines and recombinant techniques hold considerable promise.

Prevalence of leptospirosis in Belize.

In a search for leptospirosis in Belize, at least one current case was diagnosed among 11 febrile hospital patients examined in a 2-week period in mid-1984. Sera from 440 survey subjects in 6 schools, 4 urban communities and 9 rural communities were examined for leptospiral antibodies by the microscopic agglutination test. 20 of 174 (11.5%) school pupils, 19 of 88 (22%) urban community subjects and 66/178 (37%) rural community subjects were positive at titres greater than or equal to 1:50, and 7.5%, 17% and 26%, respectively, were positive at greater than or equal to 1:100. Among all groups of survey subjects, serological reactions to antigens in the Australis serogroup predominated. Up to 81% of 155 sera from cattle in various areas of Belize were positive at titres greater than or equal to 1:100. Serological reactions to serogroup Sejroe predominated (43%), followed by Pyrogenes (16%) and Australis (15%). 20 of 71 (28%) pig sera were positive at greater than or equal to 1:100, predominantly to serovars in the Australis serogroup (51%). Of 162 sheep or goat sera examined, 65 (40%) were positive at greater than or equal to 1:100, mainly to serogroup Autumnalis (25%), followed by Australis (15%).

Leptospires in vervet monkeys (Cercopithecus aethiops Sabaeus) on Barbados.

Agglutinins to Leptospira were found at titers of greater than or equal to 1:100 in 150 of 501 (29.9%) vervet monkeys (Cercopithecus aethiops sabaeus) bled within 1 mo of capture in Barbados. Including a further 34 of 145 bled within 1 yr of capture, the seropositivity prevalence was 28.5%. A further 35 monkeys (5.4%) had traces of agglutinins or gave titers of 1:50. The proportion of seropositive adults (41.5%) was more than twice that of seropositive immature monkeys (17.6%). Among adults, 49.2% of males and 35.7% of females were seropositive, while among juveniles proportions of seropositive males and females were similar (17.8% and 17.4%, respectively). Seropositivity prevalences tended to increase in proportion to rainfall. In each of 165 of the 184 positive sera, a single serogroup predominated in the serological reactions. These serogroups were Ballum (61%), Icterohaemorrhagiae (16%), Autumnalis (15%), Pyrogenes, Panama, Pomona, Tarassovi and Canicola (8% altogether). In the other 19 positive sera no single serogroup predominated. Serial bleeding showed that vervet monkeys can retain naturally-acquired antibodies to Leptospira for at least 2.5 yr. The evidence suggests that vervet monkeys in Barbados are transmitting leptospiral infections among themselves independently of other groups of animals, and are not a major source of human leptospirosis.

Leptospires in the marine toad (Bufo marinus) on Barbados.

Leptospires were isolated from the kidneys of four of 211 toads (Bufo marinus) caught on Barbados. Two of the isolates were identified as Leptospira interrogans serovar bim in the Autumnalis serogroup (the most common cause of leptospiral illness on Barbados), and two as possibly new serovars in the Australis serogroup. Sera from 198 of the toads were examined by the leptospire microscopic agglutination test. Forty-two (21%) were positive at titers of greater than or equal to 1:100, and 54 (27%) at greater than or equal to 1:50. The predominating serogroups were Australis (50%), Autumnalis (23%) and Panama (13%). The agglutination tests on the culture-positive toads showed that serologic studies alone may be of limited value in these animals. Bufo marinus can harbor pathogenic leptospires, and it may be a significant source of the Autumnalis serogroup infections in the Caribbean.

Mongoose rabies.

Mongooses are indigenous to Africa and Asia and have been introduced elsewhere. In Cuba, Puerto Rico, Grenada, and South Africa they are the major reservoir and vector of rabies. Elsewhere, sporadic cases of mongoose rabies are reported, but dog rabies can mask their importance. Population density probably determines the importance of the mongoose as a reservoir of rabies. In Grenada during a 4-year period, nearly 30% (and in some areas greater than 50%) of mongooses were found to have rabies serum neutralizing antibodies. The annual proportions of mongooses that were serum antibody-positive and virus-positive, respectively, were inversely related. Natural immunity in mongooses probably lasts for life. In contrast with foxes, in mongooses immunity is an important factor in the epizootiology of rabies. Inoculation of mongooses with Evelyn-Rokitnicki-Abelseth (ERA) vaccine induced a good immunologic response and augurs well for the success of vaccination in the field. The effects of wildlife vaccination on animal populations with high levels of natural immunity need to be investigated before costly campaigns are started.

Leptospires in Rattus spp. on Barbados.

Among 138 Rattus norvegicus and 98 R. rattus trapped on Barbados in 1964-65 and examined for evidence of leptospiral infection, seropositivity prevalence rates were similar (34 and 30%, respectively), but isolation/dark field microscopy rates were higher in R. norvegicus (27%) than R. rattus (15%). R. norvegicus carried mainly serogroup Icterohaemorrhagiae and R. rattus mainly serogroup Autumnalis. These two serogroups cause 90% of severe human leptospirosis on the island.

Leptospirosis in piggery workers on Trinidad.

Of 133 cases of human leptospirosis recorded in Trinidad between 1977 and the end of February 1982, at least eight (6%) were in people who worked on pig farms. Three of the eight died, and their presumptive infecting serogroups were Icterohaemorrhagiae (3), Canicola (2), Pyrogenes (2) and Grippotyphosa (1). Six of the eight cases were followed up. Altogether, sera from 201 pigs, 78 other livestock animals, 38 workers and 34 dogs were tested for leptospiral agglutinins. The seropositivity prevalence among pigs on farms with human illness (43% greater than or equal to 1:100) was similar to that in pigs from farms not associated with illness (46%), but the titres among the former group (geometric mean 209.5) were higher than among the latter (91.5), where only titres less than or equal to 1:400 were recorded. Similar infecting serogroups were recorded among pigs on the two groups of farms, with Icterohaemorrhagiae, Autumnalis, Canicola and Pyrogenes most frequently recorded overall. There was little evidence of the pig-adapted serogroups Pomona and Tarassovi. Twelve of 13 workers (93%) from a farm on which at least two other people had contracted leptospirosis had serological evidence of exposure, compared with seven of 24 (29%) on a neighbouring farm not associated with human illness. Dogs and rodents are thought to be the major sources of leptospirosis in pigs and piggery workers in Trinidad.

Leptospiral antibodies in patients from a Barbadian general practice.

Sera from 1,419 patients who attended a Barbadian general practice for a variety of complaints between 1 April 1984 and 30 April 1988 were examined for leptospiral agglutinins by the microscopic agglutination test (MAT). Sera from the 42 patients with pyrexia of unknown origin, jaundice or kidney involvement, and the 26 patients with titres greater than or equal to 1:400 in the MAT, were also examined by the ELISA for IgM and IgG antibodies. Current or recent mild leptospiral infection was diagnosed in four of the patients, but some mild cases may have been missed. Two further cases of leptospirosis were recorded after the study period ended; both were very ill but responded well to doxycycline therapy. Altogether 177 of the 1,419 patients (12.5%) had agglutination titres greater than or equal to 1:50. These were 104/739 males (14%) and 73/680 females (11%). Seropositivity tended to increase with age (p less than 0.01), and the highest rates were in agricultural workers (35%), labourers (24%) and non-manual outdoor workers (19%). The difference in seropositivity between the main occupational groups was highly significant (p less than 0.001). The serogroups most commonly recorded among the seropositive patients were Autumnalis (31%), Panama (26%), Australis (24%) and Pyrogenes (20%). Autumnalis predominated in each of the main occupational groups except indoor non-manual workers where Panama, Pyrogenes and Australis occurred more frequently. 95% of the positive titres ranged between 1:50 and 1:400. Titres tended to increase with age, but there was no obvious association between higher titres and particular occupations.(ABSTRACT TRUNCATED AT 250 WORDS)

The incidence of severe leptospirosis in Trinidad.

Between February 1977 and September 1982, sera from febrile patients from all areas of Trinidad were examined for leptospiral agglutinins; 158 of 1714 patients (9%) were confirmed as current cases of leptospirosis (annual average 26 or 2.6 per 100,000 population). Of the remaining 1556 patients only 711 provided paired sera; 167 of these (23%) showed evidence of previous leptospiral infection. Of the 845 single samples, 125 were positive. The true incidence of the disease is probably much higher than reported here. Males comprised 74% of the current cases; their highest rate of infection was in the 30-39 year age group, the incidence in late teenage approaching this rate. For females, incidence increased with age up to late middle-age. The presumptive infecting serogroups most commonly recorded among the 158 current cases were Icterohaemorrhagiae (37%), Canicola (16%) and Autumnalis (11%). 13 of the cases died (case fatality rate, 8.2%).

Molecular cloning of mannose-6-phosphate reductase and its developmental expression in celery.

Compared with other primary photosynthetic products (e.g. sucrose and starch), little is known about sugar alcohol metabolism, its regulation, and the manner in which it is integrated with other pathways. Mannose-6-phosphate reductase (M6PR) is a key enzyme that is involved in mannitol biosynthesis in celery (Apium graveolens L.). The M6PR gene was cloned from a leaf cDNA library, and clonal authenticity was established by assays of M6PR activity, western blots, and comparisons of the deduced amino acid sequence with a celery M6PR tryptic digestion product. Recombinant M6PR, purified from Escherichia coli, had specific activity, molecular mass, and kinetic characteristics indistinguishable from those of authentic celery M6PR. Sequence analyses showed M6PR to be a member of the aldo-keto reductase superfamily, which includes both animal and plant enzymes. The greatest sequence similarity was with aldose-6-phosphate reductase (EC 1.1.1.200), a key enzyme in sorbitol synthesis in Rosaceae. Developmental studies showed M6PR to be limited to green tissues and to be under tight transcriptional regulation during leaf initiation, expansion, and maturation. These data confirmed a close relationship between the development of photosynthetic capacity, mannitol synthesis, and M6PR activity.

A twelve-year study of leptospirosis on Barbados.

Between November 1979 and December 1991, 398 cases of severe leptospirosis were confirmed on Barbados (range for 1980-1991 23-56; mean 32.7; incidence 13.3/100,000/year). For the six-year periods 1980-1985 and 1986-1991 there was no significant change in incidence with time. Incidence is unlikely to change significantly in the next decade. Monthly average case numbers ranged from 1.4 (July) to 4.3 (November). The average (2.8) for June to December (the 7 wetter months) was not significantly higher than that (2.5) for January to May (the 5 drier months). The age range was 7-86. There were three times as many male cases (302) as female (96), and nearly 10 times as many in those < 35. Although the highest number of cases (69) was in males aged 15-24, the highest incidence was in the older age groups, particularly the male 65-74 year-olds, and the female 55-64 year-olds. Leptospirosis was the proven cause of death in 55 (13.8%) hospital patients (annual range 0-13, mean 4.5). Some of a further 39 fatalities might have been cases. Death from leptospirosis was nearly twice as common among the women as among the men. Only one patient under 20 years of age died. Leptospira were isolated and identified from 117 (29.4%) of the 398 sick patients. The infecting organisms were bim (serogroup Autumnalis--75), copenhageni (Icterohaemorrhagiae-26), arborea (Ballum-14) and bajan (Australis-2). These infecting serovars could not be distinguished clinically, but infection was milder in children than in adults. Despite its predominance in surveyed children, serogroup Panama was virtually absent in this study. Rainfall is the major factor affecting the distribution of cases; not surprisingly, sanitation workers and agricultural workers appear to be the groups at highest risk. The general lack of clear-cut risk factors reflects the ubiquity of leptospires in the environment and the fact that the disease is not entirely occupational.

Leptospires in the whistling frog (Eleutherodactylus johnstonei) on Barbados.

Two groups of whistling frogs (Eleutherodactylus johnstonei) comprising 99 and 117 animals were examined for leptospiral infection. Group I animals were caught in 14 areas of Barbados, and Group II animals in seven areas of suburban Bridgetown. Leptospires were isolated from the kidneys or body fluid of six frogs in Group I and the kidneys of 3 frogs in Group II. Two of the Group I isolates died out; the others were identified as bajan (a new serovar in the Australis serogroup) (6) and bim (Autumnalis) (1). The macerated body tissues and fluid of Group I frogs were put into phosphate buffered saline and examined by the microscopic agglutination test using 22 antigens. The results were all negative. For the Group II frogs the methodology was altered; blood was collected onto filter paper discs and allowed to dry out before being agitated in PBS and examined by the MAT. 15/117 (12.8%) animals were positive at greater than or equal to 1:100 and 19 (16.2%) at greater than or equal to 1:50. The geometric mean titre was 179. Seventeen of the sera reacted predominantly to antigens in the Australis serogroup, and two to Pyrogenes on its own. The serological results reflected the identity of the isolates. Serovars of Australis are not known to cause illness on Barbados, but bim is the commonest cause of severe leptospirosis on the island.

Mannitol Synthesis in Higher Plants : Evidence for the Role and Characterization of a NADPH-Dependent Mannose 6-Phosphate Reductase.

Mannitol is a major photosynthetic product in many algae and higher plants. Photosynthetic pulse and pulse-chase (14)C-radiolabeling studies with the mannitol-synthesizing species, celery (Apium graveolens L.) and privet (Ligustrum vulgare L.), showed that mannose 6-phosphate (M6P) and mannitol 1-phosphate were among the early photosynthetic products. A NADPH-dependent M6P reductase was detected in these species (representing two different higher plant families), and the enzyme was purified to apparent homogeneity (68-fold with a 22% yield) and characterized from celery leaf extracts. The celery enzyme had a monomeric molecular mass, estimated from mobilities on sodium dodecyl sulfate-polyacrylamide gels, of 35 kilodaltons. The isoelectric point was pH 4.9; the apparent K(m) (M6P) was 15.8 millimolar, but the apparent K(m) (mannitol 1-phosphate) averaged threefold higher; pH optima were 7.5 with M6P/NADPH and 8.5 with mannitol 1-phosphate/NADP as substrates. Substrate and cofactor requirements were quite specific. NADH did not substitute for NADPH, and there was no detectable activity with fructose 6-phosphate, glucose 6-phosphate, fructose 1-phosphate, mannose 1-phosphate, mannose, or mannitol. NAD only partially substituted for NADP. Mg(2+), Ca(2+), Zn(2+), and fructose-2,6-bisphosphate had no apparent effects on the purified enzyme's activity. In vivo radiolabeling results and the enzyme's kinetics, specificity, and distribution (in two-plant families) all suggest that NADPH-dependent M6P reductase plays an important role in mannitol biosynthesis in higher plants.

Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation.

We further investigated the role of the Arabidopsis CBF regulatory genes in cold acclimation, the process whereby certain plants increase in freezing tolerance upon exposure to low temperature. The CBF genes, which are rapidly induced in response to low temperature, encode transcriptional activators that control the expression of genes containing the C-repeat/dehydration responsive element DNA regulatory element in their promoters. Constitutive expression of either CBF1 or CBF3 (also known as DREB1b and DREB1a, respectively) in transgenic Arabidopsis plants has been shown to induce the expression of target COR (cold-regulated) genes and to enhance freezing tolerance in nonacclimated plants. Here we demonstrate that overexpression of CBF3 in Arabidopsis also increases the freezing tolerance of cold-acclimated plants. Moreover, we show that it results in multiple biochemical changes associated with cold acclimation: CBF3-expressing plants had elevated levels of proline (Pro) and total soluble sugars, including sucrose, raffinose, glucose, and fructose. Plants overexpressing CBF3 also had elevated P5CS transcript levels suggesting that the increase in Pro levels resulted, at least in part, from increased expression of the key Pro biosynthetic enzyme Delta(1)-pyrroline-5-carboxylate synthase. These results lead us to propose that CBF3 integrates the activation of multiple components of the cold acclimation response.

An investigation of some risk factors for severe leptospirosis on Barbados.

Between November 1979 and the end of December 1986 (7.17 years), 248 cases of leptospirosis were confirmed among hospital patients on Barbados (mean 35 per year; range 25-57). Considering the 235 who were greater than or equal to 15 years of age, the annual incidence of leptospirosis was 19.2/100,000 population (14.0 for all age groups). There were 173 males and 62 females, and for cases aged 15-34 leptospirosis was 9.6 times more common in men than women. Among men, incidence increased fairly steadily with age, and an even steadier increase was apparent in women up to age 64, with some decline in later years. The incidence of disease was much higher among agricultural than other workers and the non-employed. Highest case numbers were recorded in the parishes of St Michael (65 or 28%) and Christ Church (36 or 15%), though the incidence was lowest in these two parishes (13.1/100,000 and 17.4/100,000, respectively). The highest incidence rates were in St Andrew and St Joseph (50.2 and 36.1/100,000, respectively). The incidence in areas with rainfall greater than or equal to 1600 mm (32.6/100,000) was nearly twice that in areas with rainfall less than 1600 mm (17.3/100,000). There is a clear link between cases of severe disease and recent rainfall. Using 134 patients greater than or equal to 15 years of age with fever due to other illnesses as controls, a higher proportion of cases than controls came from rural areas. The risk of contracting leptospirosis was increased for all categories of manual workers relative to the group at lowest risk (non-manual indoor workers). Sugar-cane workers were five times more likely to contract leptospirosis than were non-manual indoor workers, while those whose families minded livestock were 2.5 times more likely, and those with rodents in their garden/yard were 1.8 times more likely to do so. Other risk factors examined did not show significant associations with the disease. Despite increasing mechanization and the use of more protective clothing, agricultural workers are still at high risk from leptospirosis. The annual range of cases is likely to stay much as it is in the foreseeable future.