Investigating the role of the geminal dimethyl groups of coenzyme A: synthesis and studies of a didemethyl analogue.

An analogue 2 of coenzyme A (CoA) has been prepared in which the geminal methyl groups are replaced with hydrogens. An NMR titration study was conducted and shifts in frequency of protons in the pantetheine portion of the molecule upon titration of the adenine base were observed as has been previously reported with CoA. These studies indicate that the geminal dimethyl groups are not essential for adoption of a partially folded conformation in solution. Based on 1H-1H coupling constants, the distribution of conformations about the carbon-carbon bonds in the region of the methyl deletion were estimated. The results suggest that the conformer distribution is similar to that of CoA, but with small increases in population of the anti conformers. A simple model compound containing the didemethyl pantoamide moiety was prepared and subjected to similar conformational analysis. The coupling constants and predicted conformer distribution were almost identical to that of the CoA analogue, indicating that the conformer distribution is controlled by local interactions and not influenced by interactions between distant parts of the CoA molecule. The acetyl derivative of 2 was a fairly good substrate for the acetyl-CoA utilizing enzymes carnitine acetyltransferase, chloramphenicol acetyltransferase, and citrate synthase, with 1.3- to 10-fold increased Km values and 2.5- to 11-fold decreases in Vmax. The combined results indicate that the geminal dimethyl groups of CoA have modest effects on function and minimal effects on conformation.

Frequency of Saint Louis encephalitis virus in humans from Florida, USA: 1990-1999.

Four general frequencies of human St. Louis encephalitis (SLE) virus (epidemic, focal, sporadic, and no transmission) occurred in Florida between 1990 and 1999. An epidemic with 226 clinical cases and 11 deaths was reported from 28 Florida counties between July 1990 and January 1991. During the autumn of 1993, a focal outbreak was reported from Lee (5 cases) and Collier (3) Counties in southwest Florida. During the autumn of 1997, sporadic transmission to nine humans was reported from five Florida counties (Brevard [1 case], Polk [3], Charlotte [1], Lee [2], and Palm Beach [2]). Human infection with SLE virus depends on a number of variables that drive virus transmission. These include vector, virus, and avian host abundance, and meteorological events, especially rainfall. We monitored the abundance and serological status of wild avian amplification hosts, virus isolations from Culex nigripalpus Theobald females, and SLE virus transmission to sentinel chickens during 1990, 1993, and 1997. The epidemic of 1990 was characterized by conditions that produced an unusual abundance of vector mosquitoes and avian amplification hosts early in the year. We propose that epidemics may result when a specific combination of biotic and abiotic conditions favor SLE virus minimum field infection rates that approach 1:1,000 in Cx. nigripalpus vectors.

Dengue surveillance in Florida, 1997-98.

Recent dengue outbreaks in the Caribbean and Central and South America and the presence of competent mosquito vectors increase the likelihood of future autochthonous transmission in Florida. During April 1997 to March 1998, a laboratory-based active surveillance program detected 18 cases of dengue involving all four dengue serotypes. All patients reported recent travel to countries with indigenous dengue transmission. These results demonstrate that dengue infections are imported into Florida at a much higher rate than reflected by previous passive surveillance; therefore, the risk for local dengue transmission may be increasing.

Avian serology in a St. Louis encephalitis epicenter before, during, and after a widespread epidemic in south Florida, USA.

Blood and serum from 3,915 wild and domestic birds (2,590 resident, 139 migrant, and 1,186 captive), representing 56 species collected in central Florida from 1989 through 1997, were analyzed for evidence of St. Louis encephalitis (SLE) virus transmission. All sera were tested for SLE hemagglutination inhibition (HI) antibody. Selected sera and bloods were tested for SLE neutralizing (NT) antibody and virus. The reproductive success of resident birds was highest from 1990-1992 and lowest from 1994-1997. Transmission of SLE to resident birds, especially mourning doves (Zenaida macroura), peaked during the summer of 1990, a year during which a widespread SLE epidemic was recorded in central Florida. The SLE antibody-positive resident birds 1st appeared during September of the epidemic year. Some SLE, HI antibody-positive resident birds were captured throughout 1991, but only 5% were yearlings, compared with 36% in 1990. By 1993, wild resident birds expressing HI and NT antibodies to SLE had nearly disappeared. None of the migrant birds tested were SLE-positive. Sentinel chickens maintained in Indian River County during the epidemic year seroconverted to SLE starting in early July with peak seroconversion rates in August, September, and October. High (> or = 50%) SLE seroconversion rates in sentinel chickens preceded those in wild birds by 10 wk and preceded peak human SLE transmission by at least 8 wk. Major SLE epidemics in south Florida depend on abundant wild bird populations, especially during the amplification phase of the transmission cycle. We propose that hard winter freezes along the temperature-subtropical climatic zone interface in central Florida, at approximately 27 degrees 30' North Latitude, opens foraging and nesting habitats for ground-feeding birds, resulting in high reproductive success and an abundance of seronegative individuals that rapidly amplify the SLE later in the year.

Eastern equine encephalitis transmission to emus (Dromaius novaehollandiae) in Volusia County, Florida: 1992 through 1994.

From May 1992 through October 1994, sera were collected from 204 domestic emus (Dromaius novaehollandiae) at a ranch in Volusia County, FL, and tested for antibody evidence of arboviral infection. Hemagglutination-inhibition (HI) and neutralizing (NT) antibodies to eastern equine encephalitis (EEE) virus were identified in sera collected during each year. In addition, HI and NT antibodies to St. Louis encephalitis virus were detected in 3 naturally infected emus. Isolations of EEE virus were made from emu blood and tissues collected in 1992 and 1994, when EEE-related mortality in emus was 14% and 1%, respectively. A total of 259 mosquito pools was collected and tested for arbo-viruses during the 3-year study. The EEE virus was isolated from 4 of 140 mosquito pools (2 Anopheles crucians and 2 Culex erraticus pools) in 1992 and 3 of 10 pools (all Culex nigripalpus) in 1994. Emus vaccinated against EEE virus showed evidence of short-term HI antibody acquisition. Evidence of EEE antibody transfer from naturally infected hens to their offspring is reported.

Antibodies to arthropod-borne encephalitis viruses in small mammals from southern Florida.

From 1987 through 1991, blood samples were collected from 10 species of small mammals in Indian River Country, Florida (USA). Sera from 1,347 animals were analyzed for hemagglutination-inhibition (HI) antibody to St. Louis encephalitis (SLE) and eastern equine encephalitis (EEE) viruses. Of these, 75 (5.6%) were positive for HI antibody to SLE virus and 121 (9.0%) were positive for EEE antibody. Sera from five mammalian species were tested for neutralizing (NT) antibody to SLE, EEE, Highlands J (HJ a member of the western equine encephalitis virus complex), or Everglades (EVE, a member of the Venezuelan equine encephalitis complex) viruses. By serum neutralization tests, 26 (46%) of 57 had SLE antibodies, 14 (24%) of 58 had EEE antibodies, two (3.2%) of 63 had HJ antibodies, and 9 (14%) of 63 had EVE antibodies. One Sigmodon hispidus and one Peromyscus gossypinus had NT antibodies both to EEE and HJ viruses. Blood samples from 512 mammals were tested for virus. Isolations of one EVE virus and two unidentified arenaviruses were made from P. gossypinus and one EVE virus isolate was made from a S. hispidus.

Transmission patterns of St. Louis encephalitis and eastern equine encephalitis viruses in Florida: 1978-1993.

Sentinel chickens were maintained at field sites in 40 Florida counties for varying periods between 1978 and 1993. For each county, the total number of St. Louis encephalitis virus (SLE) or eastern equine encephalitis (EEE) virus seroconversions were divided by the number of chickens exposed to calculate a mean annual seroconversion rate. These rates were used to evaluate the annual and geographical distributions of these viruses within Florida. For SLE, the rates in counties that reported human SLE cases during a widespread epidemic in 1990 were compared with adjusted mean annual seroconversion rates calculated by excluding the epidemic year seroconversion data. In general, actual rates were higher than adjusted rates in counties where human SLE cases were reported in 1990. Similar calculations were made for EEE virus that was unusually abundant in northern Florida during the spring and summer of 1991. In general, EEE virus was distributed in the panhandle and northern regions and SLE virus in the central and southern regions of the state. However, this distribution was not exclusive, and during years of high transmission, either virus can extend beyond its normal range. The annual distribution of SLE and EEE viruses was sporadic, ranging from years with little detectable activity to years with widespread, high-level transmission.

Antibodies to St. Louis encephalitis virus in armadillos from southern Florida.

From January 1990 through March 1991, 189 armadillos (Dasypus novemcinctus) were collected from Brevard and Glades Counties in southern Florida (USA). The sera were analyzed for hemagglutination-inhibition (HAI) antibodies against St. Louis encephalitis (SLE) and eastern equine encephalomyelitis (EEE) viruses. None of the armadillos had detectable HAI antibody to EEE virus, but 59 (31%) had antibodies against SLE virus. Sera from 31 of the HAI-positive armadillos contained significant levels of neutralizing (NT) antibody to SLE virus. Armadillos captured during the 1990 SLE human epidemic in south Florida had a greater prevalence of HAI and NT antibody to SLE virus than did animals captured before the start of the epidemic. This is evidence that armadillos were fed on by mosquitoes infected with SLE virus. We propose that armadillos may be involved in the SLE amplification and transmission cycles in Florida.

FACTORS AFFECTING THE RATE OF CALCIFICATION IN HALIMEDA OPUNTIA (L.) LAMOUROUX AND HALIMEDA DISCOIDEA DECAISNE(1).

Halimeda is a prominent part of the calcifying algae in the coral-reef lagoon ecosystems in the Caribbean. Experiments were performed on the Cayo Enrique Reef off Puerto Rico and in the laboratories of the University of Maryland to study factors influencing the calcification processes. Halimeda opuntia has a higher percentage of calcium carbonate than does Halimeda discoidea and a faster rate of incorporation. Halimeda opuntia and Halimeda discoidea show a stimulation of incorporation by light as well as a diurnal rhythm under identical conditions of illumination. Both phenomena parallel the rhythm of chloroplast migration within the plant. Calcification is also stimulated by the addition of carbon dioxide. Such evidence clearly indicated a light-linked mechanism which could involve photo-synthesis. However other metabolic processes, such as respiration, are also implicated. Aeration alone accelerates calcium incorporation. Nitrogen sources inhibit the incorporation of calcium during the day, indicating that cellular ammonia production is probably not responsible for precipitation. The differential wash-out rates of calcium absorbed during the day compared to those at night support the concept of a 2-step mechanism for calcification.