Highly effective, water-soluble, hemocompatible 1,3-propylene oxide-based antimicrobials: poly[(3,3-quaternary/PEG)-copolyoxetanes].

This study focuses on the solution antimicrobial effectiveness of a novel class of copolyoxetanes with quaternary ammonium and PEG-like side chains. A precursor P[(BBOx-m)(ME2Ox)] copolyoxetane was prepared by cationic ring-opening copolymerization of 3-((4-bromobutoxy)methyl)-3-methyloxetane (BBOx) and 3-((2-(2-methoxyethoxy)ethoxy)methyl)-3-methyloxetane (ME2Ox) to give random copolymers with 14-100 (m) mol % BBOx. Reaction of P[(BBOx-m)(ME2Ox)] with dodecyl dimethylamine gave the corresponding quaternary P[(C12-m)(ME2Ox)] polycation salts, designated C12-m, as viscous liquids in 100% yield. BBOx/ME2Ox and C12/ME2Ox ratios were obtained by (1)H NMR spectroscopy. C12-m molecular weights (M(n), 3.5-21.9 kDa) were obtained from (1)H NMR end group analysis. DSC studies up to 150 °C showed only thermal transitions between -69 and -34 °C assigned to T(g) values. Antibacterial activity for the C12-m copolyoxetanes was tested by determining minimum inhibitory concentrations (MICs) against Gram(+) Staphylococcus aureus and Gram(-) Escherichia coli and Pseudomonas aeruginosa . MIC decreased with increasing C12 mol percent, reaching a minimum in the range C12-43 to C12-60. Overall, the antimicrobial with consistently low MICs for the three tested pathogenic bacteria was C12-43: (bacteria, MIC, µg/mL) E. coli (6), S. aureus (5), and P. aeruginosa (33). For C12-43, minimum biocidal concentration (MBC) to reach 99.99% kill in 24 h required 1.5× MIC for S. aureus and 2× MIC for E. coli and P. aeruginosa . At 5× MIC against a challenge of 10(8) cfu/mL, C12-43 kills ≥99% S. aureus , E. coli , and P. aeruginosa within 1 h. C12-m copolyoxetane cytotoxicity toward human red blood cells was low, indicating good prospects for biocompatibility. The tunability of C12-m copolyoxetane compositions, effective antimicrobial behavior against Gram(+) and Gram(-) bacteria, and promising biocompatibility offer opportunities for further modification and potential applications as therapeutic agents.

Some observations on the sero-prevalence of heartwater and tick infestation in Zambian goats.

A survey was carried out to define the distribution of heartwater in goats that originated from six districts in communal grazing semi-arid areas of Zambia. A total of 181 samples (40.1%) out of 451 serum samples from adult goats were positive for Ehrlichia ruminantium antibodies after screening using indirect MAP-1B antigen ELISA technique with statistically significant differences (P < 0.01) between the six districts. Out of 1 036 adult goats examined for tick infestation, 105 were infested by ticks, with Amblyomma species being the most dominant tick encountered. Amblyomma variegatum, which is the vector for heartwater transmission in Zambia constituted 42.4% of the tick species, identified. The overall tick infestation rate was 10% while the tick:goat ratio was 2.1:1. Amblyomma variegatum appears to be widespread throughout the study area, as are antibodies to E. ruminantium.

A survey of trypanosomosis in Zambian goats using haematocrit centrifuge technique and polymerase chain reaction.

The incidence of trypanosomosis was determined using the haematocrit centrifuge technique (HCT) as well as polymerase chain reaction (PCR) on 120 goat blood spots on filter paper. Both techniques failed to detect a positive reaction, implying that factors such as age, healthy appearance and small sample size notwithstanding, trypanosomosis does not seem to pose a serious threat to goat health in the districts from where the animals originated.

Demonstration of erythrophagocytosis in Trypanosoma congolense-infected goats.

Anaemia is the most prominent clinical sign of trypanosomosis in domestic animals but little is known about its pathogenesis. This work investigated erythrophagocytosis as the possible cause of anaemia. Pathogenic Trypanosoma congolense (IL3000) was intravenously inoculated into six goats at 3x10(6) trypanosomes per goat. Six other goats were maintained as controls. The infection was studied for 10 weeks and parasitaemia, packed cell volume (PCV) and serum protein levels were determined. The amount of erythrophagocytosis was determined from the amount of 51Cr-labelled red blood cells (RBCs) phagocytosed by self mononuclear cells (MNCs) in vitro and by microscopically counting phagocytosed RBCs on Giemsa stained smears of incubated mixtures of RBCs and self MNCs. The infection resulted in trypanosomosis with rapid progressive anaemia and mean peaks of parasitaemia of about 3x10(3)ml(-1). In infected goats, a significant (P<0.05) mean reduction in PCV (of 37-22%) was observed starting from about 20 days up to 56 days post-infection. Within this same phase, significant (P<0.05) differences in mean radioactivity counts of (51)Cr incorporated into MNCs were observed with infected goats' samples having counts 50% higher than the control goats' samples. Microscopically, the mean number of phagocytosed RBCs in infected goats' MNCs was noted to be 80% higher (P<0.05) than that of control goats. Appreciable increases (P<0.05) in mean serum globulin levels, from 3.5 to 4.7g/dl, were observed within 3 weeks of infection. The study showed that erythrophagocytosis is an important mechanism leading to anaemia in the pathophysiology of T. congolense infection in Zambian goats.

Mechanisms of synesthesia: cognitive and physiological constraints.

Synesthesia is a conscious experience of systematically induced sensory attributes that are not experienced by most people under comparable conditions. Recent findings from cognitive psychology, functional brain imaging and electrophysiology have shed considerable light on the nature of synesthesia and its neurocognitive underpinnings. These cognitive and physiological findings are discussed with respect to a neuroanatomical framework comprising hierarchically organized cortical sensory pathways. We advance a neurobiological theory of synesthesia that fits within this neuroanatomical framework.

The use of dynamic size-sieving capillary electrophoresis and mismatch repair enzymes for mutant DNA analysis.

The detection of base pair mismatches in limiting amounts of DNA is important in the early diagnosis of cancer and other genetic diseases. The specific type and exact location of a bp mismatch in certain genes can yield information on the likelihood of a patient developing a genetic disease, as well as the severity of the disease. We demonstrate two methods of specific DNA point mutation detection and identification that involve the integration of mismatch repair cleavage enzyme analyses with dynamic size-sieving capillary electrophoresis. The mismatch repair cleavage enzymes employ the very mechanism that a cell uses in its own mismatch recognition and repair systems. One analysis employs an isothermal signal amplification process, and the other involves polymerase chain reaction (PCR) (Hoffmann-LaRoche, Nutley, NJ, U.S.A.) for amplification of the DNA. Separation of the DNA fragments using dynamic size-sieving CE yields a cleaved fragment, providing definitive evidence of a bp mismatch. The specificity and sensitivity of the assay are facilitated by the detection of fluorescently labeled DNA fragments using laser-induced fluorescence detection; picogram quantities of a target DNA can be analyzed reproducibly.

Characterization of some Theileria parva stocks from Zambia using monoclonal antibodies.

Theileria parva parasites have been isolated from different location in Zambia where malignant theileriosis has been recorded. A total of 16 bovine lymphocytic cell lines infected with T. parva schizonts were characterized using a panel of anti-schizont monoclonal antibodies (MAbs). Comparison of the Theileria stocks isolated before (old) and after (new) the Muguga cocktail of T. parva from Kenya was used to vaccinate cattle against theileriosis in Zambia revealed differences in their reactivity against MAbs. The new isolates are showing MAb profiles similar to that exhibited by the Muguga cocktail which was used to vaccinate cattle in these areas between 1983 and 1989. These results suggest that the use of the Muguga cocktail to vaccinate animals against theileriosis in Zambia may have introduced Theileria stocks of different antigenic properties.

RecA-assisted rapid enrichment of specific clones from model DNA libraries.

An approach to library screening is being developed, in which the desired clone is "fished" out of a mixture of all the recombinants in a library with a RecA-coated probe. In the current embodiment of this method, we used as a probe the (+) strand of an M13 phage containing a fragment of the human albumin gene and a (dA)49 stretch. We screened a library of two plasmids, one containing the same albumin fragment as the probe, and one heterologous to the probe in 50-100 fold molar excess. The plasmids were linearized. Probe and library were reacted in the presence of RecA, the mixture was loaded onto an oligo(dT) column, which retained the probe-target complex by base-pairing to the dAs of the probe, the uncaptured plasmids were washed, and the probe-target complex was released from the column, religated and propagated into E. coli. Recovery of the homologous target was 15-28%, and enrichment for the homologous plasmid was 200 to 400-fold. This approach may provide a general method for expedited DNA library screening.

Prostaglandin E2 down-regulates the expression of HLA-DR antigen in human colon adenocarcinoma cell lines.

Prostaglandins (PG) have been implicated in the pathogenesis of cancer and play an important role in immune regulation. Colon cancer is associated with elevated levels of PGE2, while aspirin, the prototypical inhibitor of PG synthesis, appears to reduce the incidence of colon cancer by 50%. We have observed that in human colon cancer the expression of HLA class I and II antigens is reduced or lost; loss of HLA antigens is suspected to be a mechanism by which the malignant cell escapes the immune surveillance. We investigated the effect of these eicosanoids on the expression of HLA antigens in human colon adenocarcinoma cell lines. PGE2 down-regulated the expression of the class II antigen HLA-DR in SW1116 cells (65% reduction at 2.8 x 10(-8) M). This effect was dose- and time-dependent, reversible, and specific (PGF2 alpha and LTB4 had no effect; the expression of carcinoembryonic antigen and class I genes were not affected). Aspirin induced the expression of HLA-DR in HT29 cells, a cell line not expressing constitutively HLA-DR. The reduction of HLA-DR by PGE2 was accompanied by reduced messenger RNA (mRNA) levels of HLA-DR alpha and reduced transcription of the corresponding gene. In contrast to HLA-DR, none of these three eicosanoids affected the expression of HLA class I genes, as assessed via determination of protein expression by fluorescence flow cytometric analysis and evaluation of the corresponding class I mRNA levels. We conclude that PGE2 specifically down-regulates the expression of HLA-DR, while it does not affect the expression of class I antigens.(ABSTRACT TRUNCATED AT 250 WORDS)

Memory for brief, widely spaced odor presentations in the rat.

Rats trained on a series of 16 novel 2-odor discrimination tasks using a 10-s intertrial interval (ITI) rapidly improved in performance and made only 0-3 errors by the end of the test series. They were then tested on other novel pairs of odors, but with a 10- and a 30-min interval between trials. There was no decrement in performance accuracy in the longer ITI tests and, in most cases, criterion performance was achieved after making zero or 1 error after the first (information) trial. These results demonstrate that rats have the capacity to remember for at least 30 min whether a single brief presentation of a novel odor was followed by a reward.

Lithocholic acid inhibits the expression of HLA class I genes in colon adenocarcinoma cells. Differential effect on HLA-A, -B and -C loci.

Loss of HLA antigen expression is considered to be one of the mechanisms whereby tumor cells escape immune surveillance. We recently observed reduced or lost expression of HLA antigens during human colon carcinogenesis. We studied the effect of bile acids (BAs), long implicated in the pathogenesis of colon cancer, on the expression of HLA class I antigens in human colon adenocarcinoma cells. Lithocholic acid (LCA) decreased by 42% the expression of HLA class I antigens on the surface of these cells. This dose-dependent reduction was specific for both the target genes and the chemical structure of LCA, and was not evident in cultured liver cells. None of the other BAs that were tested manifested this effect. LCA, and to a lesser extent deoxycholic acid (DCA), decreased steady-state HLA class I mRNA levels. LCA decreased the rate of transcription of HLA-B (64%) and HLA-C (87%) but not HLA-A; DCA had a similar but less pronounced effect. In transient gene expression (CAT assays) experiments, we evaluated the role of a 0.6-0.7 kb EcoRI/XbaI sequence from the 5' flanking region of HLA-A2, -B7 and -Cw7 genes in the regulation of class I gene expression by LCA. LCA down-regulated by 70% the expression of the reporter gene for all three genes. We interpret these results as indicating a differential regulation of the three HLA loci by LCA. Our findings, demonstrating a profound effect of LCA on HLA class I gene regulation, raise the possibility that such a mechanism may be operative in vivo.

Intrinsic resistance of cervical squamous cell carcinoma cell lines to methotrexate (MTX) as a result of decreased accumulation of intracellular MTX polyglutamates.

The causes of intrinsic of intrinsic MTX resistance in four human cervical squamous cell carcinoma cell lines (SiHa, C-33A, ME-180, C-4I) with different sensitivities to methotrexate (MTX) were determined. The in situ or whole-cell assay for thymidylate synthase (TS) was used to screen for known causes of MTX resistance, including increased dihydrofolate reductase (DHFR), altered DHFR, impaired MTX uptake, and decreased formation of MTX polyglutamates. While all four cell lines displayed initial sensitivity to MTX as demonstrated by a TS activity of < 20% following a 3-hr incubation with MTX, TS activity recovered in three of the four cell lines following a 4-hr incubation in drug-free media. Determination of MTX-polyglutamate accumulation in the four cell lines following a 24-hr incubation with 10 microM [3H]MTX revealed that a higher total intracellular level of MTX polyglutamates was achieved in the sensitive cell line (SiHa), with 54% occurring as long-chain tri-, tetra-, or pentaglutamates. The three resistant cell lines were found to contain less total MTX polyglutamates, with only 38, 14, and 13% occurring as long-chain MTX polyglutamates. Intrinsic MTX resistance in some cervical squamous cell carcinoma cell lines appears to be associated with a diminished accumulation of long-chain MTX polyglutamates, which are preferentially retained intracellularly.

Spinal cord mensuration--a comparative study on the spinal cord segments and spinal nerves in Zambian goats.

It has been known that the relative length of spinal cord and its segmental volume in domestic animals has established that the dynamics of spinal cord is directly related with the functions of the limbs and in particular to their feeding habits. Bilateral rostrocaudal measurements of spinal nerves involving their root attachment length, root emergence length, interroot length, segment length and cross sectional area were recorded on the dorsal and ventral surfaces of each segment of the spinal cords of five local healthy Zambian goats. We identified that the brachial and lumbar enlargements have involved identical number of spinal cord segments. Brachial and lumbar enlargements extended from C6 to T1 and L4 to L7. The average length of spinal cord was 59.9 cm and it extended up to caudal end of 5th sacral vertebrae. The root emergence length appeared to decrease gradually from C2 segment, which remained less variable in thoracic and lumbar segments and then receded sharply through sacral segments. The dorsal nerves entered spinal cord over a greater area than ventral because of more spinal rootlets. The greatest segment length lied in mid cervical region and then from lumbar segment it decreased sharply up to the end of sacral segments. It is concluded that these goats have a feeding habit similar to that of cattle rather than resting their forelimbs on the shrubs while nibbling the leaves as recorded in Asian goats. It also confirmed that the shrubs were more drooping along with grasses in the Gwembe Valley of Zambia.

Aflatoxin levels in foodstuffs in Fiji and Tonga islands.

Fungal growth is a major problem of food storage in humid environments, as occur in South Pacific countries for parts of the year. Major crops, including edible nuts, copra and root crops, are susceptible to Aspergillus growth and therefore potential contamination with aflatoxin. Liver cancer occurs in Fiji and Tonga, with the occurrence in Fijians being significantly higher than in the Indian population. Thirty-three peanut samples from farmers were analysed for aflatoxin and 50% of the samples from Fiji were positive but only 9% from Tonga, reflecting different storage practices. Local copra, cassava, and maize samples were found contaminated, with only the maize at a serious level. Twenty-five plate food samples from Fiji showed low contamination. When starch foods from the Fijian diet left after cooking were analysed to follow potential aflatoxin development only sweet potatoes showed some contamination.

Effect of Ca on Na-D-glucose cotransport across isolated renal brush-border membranes.

Brush-border membrane vesicles were prepared from rat kidney cortex by Mg precipitation. Using quin2 (free acid), intravesicular [Ca2+] was found to be 44 microM and less than 300 nM when vesicles were incubated in 0.2 mM CaCl2 or Ca-free buffer, respectively. In Ca-loaded vesicles, the initial D-glucose uptake, measured in the presence of 150 mM Na+ and 0.1 mM D-glucose inward gradients, was reduced to 30% of the control uptake. This reduction persisted when the extra-vesicular Ca2+ was chelated by ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid but was abolished in the presence of saturating concentrations of D-glucose. Whereas KG0.5 (g) for D-glucose at constant [Na] in the Ca-loaded membranes increased by approximately 50% of the control value (0.5 +/- 0.1 mM), no significant change in Jmax was observed. In contrast, both Jmax and KG0.5 (Na) for glucose, measured as a function of [Na] in the extravesicular fluid, were found to be significantly reduced. Na uptake, determined in the presence of 0.5 mM amiloride, was found to increase by approximately 30% of the value for control membrane. This increase was abolished when vesicles were preincubated with 0.5 mM neomycin or 0.5 mM phlorizin. The results suggest that the effect of Ca2+ on Na entry may be mediated in part by activation of phospholipase C and are consistent with a model of cotransport in which Ca2+ increases the mobility of the binary Na-sugar-translocator complex, thus leading to uncoupling of Na transport from glucose uptake ("slipping") and in part with Ca-induced Na entry by nonmediated leakage.

The effects of homogeneous human prealbumin on in vitro and in vivo immune responses in the mouse.

A highly purified preparation of human prealbumin was shown to potentiate the sensitivity of rosette spleen forming cells of adult thymectomized mice to azathioprine in vitro and in vivo and to induce the appearance of the Thy 1, 2 antigen in vitro on spleen cells of adult thymectomized mice. Prealbumin also enhanced IgM antibody synthesis to sheep red blood cells (SRBC) in vitro in 12 week old mice and in vivo in aged (45 - 58 week old) and nude (nu/nu) mice. In vivo administration, to mice that had been pre-treated with hydrocortisone, resulted in a decrease in the specific activity of thymocyte terminal deoxynucleotidyl transferase. The data indicate that the prealbumin molecule possesses immunopotentiating properties in a number of in vitro and in vivo immunocompromised murine models and that the immuno-enhancing properties of the partially purified preparation previously described were in fact due to the prealbumin component and not to other contaminating proteins.

Regulation of mitochondrial malic enzyme synthesis in mouse brain.

In a previous study [Bernstine, E.G. (1979) J. Biol. Chem. 254, 83-87] it was shown that inbred strains of mice fall into two classes based on the specific activity of mitochondrial malic enzyme [L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40] in brain. In this report we demonstrate differences between high- and low-activity strains in the development of enzyme activity levels in adult mice and show that the rate of enzyme synthesis quantitatively accounts for the inherited level of the brain enzyme. Genetic analysis has established that the locus controlling the amount of enzyme in brain (Mdr-1) is located on chromosome 7. Its linkage to Hbb and c places it in the same region of the chromosome as Mod-2, the structural gene for mitochondrial malic enzyme. By making use of deletions and a duplication that include Mod-2, evidence for cis action of Mdr-1 was obtained.