Chemical characterization of territorial marking fluid of male Bengal tiger, Panthera tigris.

The territorial marking fluid of the male Bengal tiger, Panthera tigris, consists of a mixture of urine and a small quantity of lipid material that may act as a controlled-release carrier for the volatile constituents of the fluid. Using gas chromatography and gas chromatography-mass spectrometry, 98 volatile compounds and elemental sulfur were identified in the marking fluid. Another 16 volatiles were tentatively identified. The majority of these compounds were alkanols, alkanals, 2-alkanones, branched and unbranched alkanoic acids, dimethyl esters of dicarboxylic acids, gamma- and delta-lactones, and compounds containing nitrogen or sulfur. Several samples of the marking fluid contained pure (R)-3-methyl-2-octanone, (R)-3-methyl-2-nonanone, and (R)-3-methyl-2-decanone, but these ketones were partly or completely racemized in other samples. The gamma-lactone (S)-(+)-(Z)-6-dodecen-4-olide and the C(8) to C(16) saturated (R)-gamma-lactones and (S)-delta-lactones were present in high enantiomeric purities. The chiral carboxylic acids, 2-methylnonanoic acid, 2-methyldecanoic acid, 2-methylundecanoic acid, and 2-ethylhexanoic acid were racemates. Cadaverine, putrescine, and 2-acetylpyrroline, previously reported as constituents of tiger urine, were not detected. The dominant contribution of some ketones, fatty acids, and lactones to the composition of the headspace of the marking fluid suggests that these compounds may be important constituents of the pheromone. Although it constitutes only a small proportion, the lipid fraction of the fluid contained larger quantities of the volatile organic compounds than the aqueous fraction (urine). The lipid derives its role as controlled-release carrier of the chemical message left by the tiger, from its affinity for the volatiles of the marking fluid. Six proteins with masses ranging from 16 to 69 kDa, inter alia, the carboxylesterase-like urinary protein known as cauxin, previously identified in the urine of the domestic cat and other felid species, were identified in the urine fraction of the marking fluid.

Use of a histone H4 promoter to drive the expression of homologous and heterologous proteins by Penicillium funiculosum.

Two genes encoding histone H4 (H4.1 and H4.2) from Penicillium funiculosum have been cloned and characterised. Structurally, the histone H4.1 gene is divergently linked to the histone H3 gene and the two genes are separated by approximately 800 bp. The transcription of the histone H4.1 and H4.2 genes in P. funiculosum appears to be distinctively regulated. Histone H4.1 mRNA showed a high steady-state level during the early stages of batch culture that decreased as growth reached the stationary phase. In contrast, the expression of the histone H4.2 gene was lower than that of H4.1 throughout batch growth and increased gradually with time. In order to expand the industrial application of P. funiculosum as a host for the production of heterologous proteins, the promoter of the histone H4.1 gene was successfully used to drive the expression of an intracellular bacterial enzyme, beta-glucuronidase, and a secreted homologous enzyme, xylanase C. The constitutive secretion of xylanase C was achieved in the absence of other xylanases by batch fermentation in the presence of glucose.

A modular esterase from Penicillium funiculosum which releases ferulic acid from plant cell walls and binds crystalline cellulose contains a carbohydrate binding module.

An esterase was isolated from cultures of the filamentous fungus Penicillium funiculosum grown on sugar beet pulp as the sole carbon source. The enzyme (ferulic acid esterase B, FAEB) was shown to be a cinnamoyl esterase (CE), efficiently releasing hydroxycinnamic acids from synthetic ester substrates and plant cell walls, and bound strongly to microcrystalline cellulose. A gene fragment was obtained by PCR using partial amino-acid sequences obtained from the pure enzyme and used to a probe a P. funiculosum genomic DNA library. A clone containing a 1120-bp ORF, faeB, was obtained which encoded a putative 353-residue preprotein including an 18-residue signal peptide, which when expressed in Eschericia coli produced CE activity. Northern analysis showed that transcription of faeB was tightly regulated, being stimulated by growth of the fungus on sugar beet pulp but inhibited by free glucose. The faeB promoter sequence contains putative motifs for binding an activator protein, XLNR, and a carbon catabolite repressor protein, CREA. FAEB was comprised of two distinct domains separated by a 20 residue Thr/Ser/Pro linker region. The N-terminal domain comprised 276 amino acids, contained a G-X-S-X-G motif typical of serine esterases, and was shown to be a member of a family comprising serine esterases, including microbial acetyl xylan esterases, poly (3-hydroxyalkanoate) depolymerases and CEs, and proteins of unknown function from Mycobacterium spp. and plants. The C-terminal domain comprised 39 amino acids and closely resembled the family 1 cellulose binding carbohydrate-binding modules (CBM) of fungal glycosyl hydrolases. This is the first report of a fungal CE with a CBM.

Effect of hormone variations and other factors on symptom severity in women with dystonia.

OBJECTIVE: To determine if any relationship exists between the severity of symptoms in women with dystonia and female reproductive hormonal variations. PATIENTS AND METHODS: We surveyed 279 women with dystonia seen at Mayo Clinic Scottsdale over a 6-year period (1990-1995), and 204 responded. The women were asked questions regarding their reproductive and menstrual histories and dystonia severity and other questions with an emphasis on possible exacerbating or relieving factors. RESULTS: Although in the majority of women hormonal influences had no consistent effect on dystonia symptom severity, 26 (41.9%) of 62 premenopausal women noted a change in the severity of their dystonic symptoms in relation to the 3 phases of their menstrual cycle. Other factors that exacerbated dystonia included stress and fatigue, while sleep improved symptoms. Pregnancy, menopause, and hormone replacement therapy had no effect on symptoms. CONCLUSIONS: Menstrual cycling may result in subjective worsening of dystonia symptoms in some women with dystonia. Further clinical and physiologic evaluation is indicated in such patients, as they may represent an important subgroup of dystonic patients that might yield some clues to the pathophysiology of dystonia and to improved treatment strategies.

Barium enema: detection of colonic lesions in a community population.

OBJECTIVE: The purposes of this study were to assess the prevalence of colonic lesions detected at barium enema in a community practice, to compare the findings at barium enema between patients who are asymptomatic and have no known risk factors for colorectal cancer (screening group) and patients who have symptoms of colonic disease or have known risk factors, and to determine if a questionnaire about symptoms and risk factors is an appropriate screening tool. SUBJECTS AND METHODS: A self-administered questionnaire about colorectal symptoms and risk factors was given to 1779 patients scheduled for barium enema examination. On the basis of their responses, patients were divided into three groups: screening group (asymptomatic, without risk factors), symptomatic, and asymptomatic with risk factors. Each patient underwent a fluoroscopic barium enema. We then compared the results (number, histologic type, size of lesion(s), location in the colon, and Patient's age) and risk factors among the three groups. RESULTS: At least one lesion within the colorectum was found in 166 (9%) of 1779 patients at combined proctosigmoidoscopy and barium enema. The prevalence of lesions in the 111 patients with at least one lesion above the rectum at barium enema was 4% (32 of 738) for the screening group, 8% (38 of 476) for asymptomatic patients with risk factors, and 7% (41 of 565) for symptomatic patients (p = .015 when comparing the prevalence in the screening group with the prevalences in the other two groups). Twenty-nine percent of all colonic lesions were found in the screening group. Among the asymptomatic patients, risk factors that included a history of colorectal polyps and advanced age were associated with a significantly higher prevalence of colonic polyps found at barium enema. In the symptomatic group, if patients with histories of polyps were excluded, we were unable to identify other risk factors that led to a significantly higher prevalence of polyps. CONCLUSION: Asymptomatic patients without known risk factors have a significantly lower prevalence of colonic polyps than either symptomatic patients or patients with risk factors alone. Despite this lower prevalence, 29% of all lesions in our series were in the screening group. Assessment of risk factors through a patient questionnaire was not helpful in identifying a group of patients with a higher prevalence of lesions--except for a history of polyps. Management decisions based on a patient questionnaire should be approached with caution. When low-risk patients are denied screening examinations, a significant number of lesions will be missed.

Central venous oxygen saturation, arterial base deficit, and lactate concentration in trauma patients.

Our object was to explore the usefulness of central venous oxygen saturation, arterial base deficit, and lactate concentration in the evaluation of trauma patients. In busy urban trauma centers, limited operating room availability may necessitate that certain hemodynamically stable patients experience some delay between diagnosis of injury and surgery. Because hemodynamic compromise may occur before operation is undertaken, some means of identifying those patients who have the most severe injuries or who are at greatest risk for hemodynamic instability would be useful. We prospectively studied 40 patients with operative truncal injuries admitted to the Cook County Trauma Unit, Chicago, to examine the usefulness of postresuscitation central venous oxygen saturation (ScvO2), arterial lactate concentration, and arterial base deficit in this regard. Preoperative hypotension occurred in 12.5 per cent of these initially stable patients. ScvO2 did not significantly correlate with any of the parameters of blood loss and severity of injury examined. However, both base deficit and lactate concentration correlated with transfusion requirements; in addition, base deficit correlated with trauma score, and lactate correlated with peritoneal shed blood volume. Our data suggest that, after resuscitation, arterial base deficit and lactate concentration may be better indicators of blood loss than is ScvO2.

[A method for assessing the microfloral status of the human intestines by the quantity of adhesion-active bacteria and by the type of adhesins]. / Sposob otsenki sostoianiia mikroflory kishechnika cheloveka po kolichestvu adgezivno-aktivnykh bakterii i tipu adgezinov.

The method for the evaluation of the state of intestinal microflora in the diagnosis of enteric diseases by the determination of the number of actively adhesive bacteria and the type of adhesins in the primary culture of human microflora has been developed. This method permits the simultaneous analysis of all colonies grown in a given Petri dish by the sign of their adhesion, which eliminates the possibility of mistakes due to the occasional choice of colonies, as it may happen in the agglutination test. The time necessary for the study of adhesive properties is reduced, as the result is evaluated as early as on day 2 after the inoculation of the seed material. It is also of importance that the method is simple in realization, needs no special equipment, preparation and staining of the material. To determine the type of adhesins, a series of identical inoculations may be obtained by replication with the use of red blood cells of different animals. The proposed method may be used for the confirmation of the etiological role of enterobacteria as evidenced by the presence of high percentage of their actively adhesive colonies.

[The role of adhesin in the polymeric structure of Vi-antigen]. / Rol' adgezina v polimernom stroenii Vi-antigena.

Samples of Vi-antigen subjected to hydrolysis of different duration have been studied. As revealed in this study, 12-hour hydrolysis causes the depolymerization of the preparation. The fragments thus obtained have been found to possess Vi-specificity. The polymeric form of Vi-antigen contains adhesin differing in specificity from Vi-determinant. This component is absent in the low-molecular fraction of Vi-antigen. Adhesin is the binding element of the molecule of Vi-antigen and ensures its polymeric structure.

[Protective properties of Vi-antigen preparations as dependent on their adhesin content]. / Protektivnye svoistva preparatov Vi-antigena v zavisimosti ot soderzhaniia v nikh adgezina.

Adhesins contained in the preparation of Vi-antigen have been found to enhance its immunogenic and protective properties. In the preparations of Vi-antigen obtained from Salmonella typhi and Citrobacter freundii the presence of two antigenic determinants has been revealed. One of them is associated with the Vi-receptor and the other determinant, with adhesin. Both determinants take part in the protection of mice from Salmonella infection.

[Use of the coagglutination reaction of yeast-like Candida maltosa fungi for detecting fimbriae in intestinal bacteria]. / Ispol'zovanie reaktsii koaggliutinatsii drozhzhepodobnykh gribov Candida maltosa dlia vyiavleniia fimbrii u kishechnykh bakterii.

The capacity of nonpathogenic yeast-like C. maltosa strains to coagglutinate Escherichia coli has been studied. C. maltosa cells have also been shown to coagglutinate E. coli possessing mannose-sensitive adhesins in a wide range of their concentrations (5-140 bacterial cells per C. maltosa cell). Strains belonging to types CFA/I and CFA/II with fimbriae, similarly to their corresponding paired genetically related strains without these adhesins, are practically incapable of agglutinating C. maltosa cells, while strains K88 and B41 react with them. The reaction occurs at a concentration of 9.5-37.0 and 38.0-55.5 bacteria respectively per C. maltosa cell and is not inhibited by 1% d-mannose. The suggestion that C. maltosa cell surface glycoproteins contain not only receptors for E. coli fimbriae, type I, but also components similar in their structure to receptors specific to the mannose-resistant adhesins of strains K88, K99 and 41, has been confirmed by hemagglutination inhibition with C. maltosa surface antigens as inhibiting agents.

Plant protoplast fusion.

It is now possible to regenerate plants from protoplasts of a wide range of species. As a result, genetic manipulation by protoplast fusion in vitro is now a realistic proposition. This area is exciting because protoplasts from different origins can be fused together to form new genome combinations that cannot be obtained by conventional means. Thus, protoplast fusion can be used to introduce novel germplasm into breeding programs. Practical examples include the introduction of resistance to potato leaf roll virus from the wild species Solarium brevidens by fusion with dihaploid potato (Solatium tuberosum) protoplasts (1) and the production of novel hybrids between tomato (Lycopersicon esculentum) and the sexually incompatible wild species (Solarium rickii) (2).

Plant tissue culture.

Plant tissues normally grow in an organized fashion in which specific cell types differentiate from nonspecialised meristematic cells. Plant developmental processes can be modified by culture in vitro in a suitable nutrient medium and with the application of plant growth regulators. The interactions of the main growth regulators can be complex, but at the simplest level auxins can cause cell enlargement and division, cytokinins cause cell division, gibberellins cause elongation, and abscisic acid inhibits growth (1). When meristems are cultured, it is possible to maintain organization and multiply such meristems, and this is the basis of micropropagation. If a suitable combination of growth regulators is chosen, however, cultured tissues can be grown in a disorganized and undifferentiated way to form callus. Single isolated wall-less cells, protoplasts, can also be released from tissues following enzymatic degradation of cell walls, and these can be cultured to form callus. In both cases the aim is to regenerate intact plants from disorganized callus, usually by manipulating growth regulator concentrations so that the cytokinin/auxin ratio is increased (2).

Direct gene transfer into plant protoplasts.

The techniques of plant molecular biology have advanced rapidly in the last 5 yr, and, for a number of plant species, including some crops, it is now possible to bypass traditional plant breeding techniques and introduce specific genes directly. The earliest reports of the transformation of plants with foreign genes exploited the fact that the soil bacterium Agrobacterium tumefaciens, which induces the formation of crown galls, transfers part of a plasmid (the tumor-inducing, Ti, plasmid) into its host (e.g., ref. 1). The isolation and modification of this plasmid by the insertion of one or more structural genes together with regulatory elements has provided the means of genetically manipulating intact plants, cultured plant tissues, and protoplasts.

Resistance to potato leaf roll virus and potato virus Y in somatic hybrids between dihaploid Solanum tuberosum and S. brevidens.

Many somatic fusion hybrids have been produced between a dihaploid potato Solanum tuberosum and the sexually-incompatible wild species S. brevidens using both chemical and electrical fusion techniques. S. brevidens was resistant to both potato leaf roll virus (PLRV) and potato virus Y (PVY), the viruses being either at low (PLRV) or undetectable (PVY) concentrations as determined by enzyme-linked immunosorbent assay (ELISA). The S. tuberosum parent was susceptible to both viruses. A wide range of resistance, expressed as a decrease in virus concentration to both viruses was found amongst fusion hybrids, four of which were especially resistant. The practicality of introducing virus resistance from S. brevidens into cultivated potatoes by somatic hybridisation is discussed.

Production of somatic hybrids by electrofusion in Solanum.

Conditions are described for large scale electrofusion of mesophyll protoplasts of dihaploid S. tuberosum with those of diploid S. brevidens. Overall fusion frequencies of 20%-30% were achieved, and following fusion, large numbers of protoplast-derived calli were obtained. Putative somatic hybrid plants were selected from the regenerated shoots by examining their morphological characteristics. Twenty-one somatic hybrids were confirmed by isoenzyme analysis and six somatic hybrids were further confirmed by Southern hybridization. Tetraploid hybrids were obtained, but cytogenetic studies indicated that more of the regenerated hybrids were hexaploid than had previously been found following chemical fusion of the same partners. Some advantages of electrofusion over chemical fusion are discussed.

Field assessment of dihaploid Solatium tuberosum and S. brevidens somatic hybrids.

Following both chemical and electrical fusion of protoplasts of a dihaploid line of potato (Solanum tuberosum), (PDH40), with those of the wild species, Solanum brevidens, 11 and 40 somatic hybrid plants, respectively were obtained. Fifteen of these somatic hybrid genotypes and the two parents were studied further in a small field trial to assess field performance and phenotypic variability. In the UK, somatic hybrid plants are classified as genetically engineered organisms, and the UK Advisory Committee on Genetic Manipulation have imposed various restrictions on field experiments. Examination of the somatic hybrids in the field showed extensive phenotypic variability, and no two genotypes were identical. Some of the variation reflected changes in chromosome numbers, but other factors were also involved. Half the somatic hybrid genotypes produced tubers in the field, although the tubers were smaller and differed morphologically from those of PDH40. The results of the study suggest that the extent of somaclonal variation manifested in somatic hybrids is greater than that found in protoplast-derived plants of potato. The implications of this observation and the current regulations concerning field experiments of somatic hybrid plants in the UK are discussed.