Symptomatology and morphology of Claviceps cyperi on yellow nut sedge in South Africa.

Symptoms of ergot on yellow nut sedge, germination of sclerotia of the causal organism, Claviceps cyperi, and morphology of fresh specimens of the pathogen are described for the first time. The initial symptom of infection was a black sooty layer on inflorescences of infected plants due to colonization of the ergot honeydew by Cladosporium cladosporioides. Sclerotia of C. cyperi started to develop in March and April and could be discerned as small protuberances on inflorescences in the place of seed. Mature sclerotia were purplish-black. They generally remained viable for less than a year and germinated without prior cold treatment, although exposure for 21 d to 5 C before incubation significantly increased the germination rate. Under moist conditions at 24 C in the laboratory, germination commenced within 4-8 wk. Stromata took about 12 d to mature. Mature capitula were distinctly lobulate with a perithecium embedded in each lobe and a collar-like appendage around the base. Although dimensions of sclerotia, stipes, capitula, asci and ascospores were larger than in the original description, the general morphology supports treatment of C. cyperi as a distinct species.

Cell volume regulation: osmolytes, osmolyte transport, and signal transduction.

In recent years, it has become evident that the volume of a given cell is an important factor not only in defining its intracellular osmolality and its shape, but also in defining other cellular functions, such as transepithelial transport, cell migration, cell growth, cell death, and the regulation of intracellular metabolism. In addition, besides inorganic osmolytes, the existence of organic osmolytes in cells has been discovered. Osmolyte transport systems-channels and carriers alike-have been identified and characterized at a molecular level and also, to a certain extent, the intracellular signals regulating osmolyte movements across the plasma membrane. The current review reflects these developments and focuses on the contributions of inorganic and organic osmolytes and their transport systems in regulatory volume increase (RVI) and regulatory volume decrease (RVD) in a variety of cells. Furthermore, the current knowledge on signal transduction in volume regulation is compiled, revealing an astonishing diversity in transport systems, as well as of regulatory signals. The information available indicates the existence of intricate spatial and temporal networks that control cell volume and that we are just beginning to be able to investigate and to understand.

Hypertonicity-induced alkalinization of rat hepatocytes is not involved in activation of Na+ conductance or Na+,K+-ATPase.

We investigated whether cell alkalinization via activation of Na+/H+ exchange is involved in the stimulation of Na+ conductance and Na+,K+-ATPase in rat hepatocytes under hypertonic stress. Osmolarity was increased from 300 to 400 mOsm/l at constant extracellular pH (7.4), whereas osmotically induced cell alkalinization (0.3 pH units in HCO3-free solutions) was mimicked by increasing extracellular pH from 7.4 to 7.8 in normosmotic solutions. In intracellular recordings with conventional and ion-sensitive microelectrodes, hypertonic stress led to a transient shift in the voltage response to low Na+ solutions (95% in exchange for choline) by -4.3 +/- 0.8 mV and a continuous increase in cell Na+ from 13.7 +/- 1.8 to 18.6 +/- 3.0 mmol/l within 8 min. In the presence of 10(-5) mol/l amiloride, these effects were reduced by 80 and 90%, respectively. In contrast, increasing pH did not change the voltage responses to low Na+ or cell Na+ concentrations significantly. In addition, application of 2 mmol/l Ba2+ pulses revealed that a sustained membrane hyperpolarization of 15.6 +/- 1.4 mV following intracellular alkalinization exclusively reflects an increase in K+ conductance. Increasing osmolarity at pH 7.4 augmented ouabain-sensitive 86Rb+ uptake from 5.5 +/- 1.1 to 8.5 +/- 1.6 nmol mg protein(-1) min(-1). In normosmotic solution at pH 7.8, 86Rb+ uptake equalled 4.9 +/- 1.6 nmol mg protein(-1) min(-1), which is not significantly different from control. We conclude that, in rat hepatocytes, cell alkalinization under hypertonic stress is not responsible for the activation of Na+ conductance and probably does not participate in the stimulation of Na+,K+-ATPase.

Activation of a Cl(-)-conductive pathway in primary cultures of rat inner medullary collecting duct (IMCD) cells under hypotonic stress.

In intracellular recordings with conventional microelectrodes on rat IMCD cells, we find that hypotonic stress depolarizes membrane voltage and decreases cell input resistance. Ion substitution experiments reveal that these effects are largely due to the activation of a prominent Cl- conductance. After block of this conductance with dideoxyforskolin a smaller concomitant increase in K+ conductance becomes detectable.

High-level expression of Na+/D-glucose cotransporter (SGLT1) in a stably transfected Chinese hamster ovary cell line.

The coding region of the high affinity Na+/d-glucose cotransporter (SGLT1) was inserted into the eukaryotic expression vector GFP-N1 under the control of a CMV promoter. The plasmid was then stably transfected into a Chinese hamster ovary cell line (CHO). Transcription and synthesis of SGLT1 were proved by Northern and Western blot analyses. Transport activities of the transfected cells (G6D3) were examined by measuring the sodium-dependent uptake of alpha-methyl[14C]d-glucoside (AMG). Kinetic analysis revealed a Vmax of 10.3 nmol/min/mg (total cell protein) and a Km of 0.26+/-0.09 mM, respectively. The concentration of phlorizin required to inhibit AMG uptake by 50% in the presence of 0.1 mM AMG was 2.35+/-1.84 microM. Electrophysiological studies showed that AMG induces a significant depolarization of membrane voltage in stably transfected CHO cells, suggesting an electrogenic Na-AMG symport. Immunoprecipitation with an antipeptide antibody yielded a nearly homogeneous polypeptide with a molecular mass of about 72 kDa. The amount of SGLT1 present in the CHO cell plasma membranes represents at least 1% of membrane protein, which is about 30-100 times higher than in natural sources, such as renal brush border membranes. In conclusion, the stably transfected G6D3 cells with a markedly high SGLT1 expression can serve as a promising model for studying cellular events related to Na+/d-glucose cotransport and for analyzing the structure and function of the cotransporter itself.

Hypertonic stress increases the Na+ conductance of rat hepatocytes in primary culture.

We studied the ionic mechanisms underlying the regulatory volume increase of rat hepatocytes in primary culture by use of confocal laser scanning microscopy, conventional and ion-sensitive microelectrodes, cable analysis, microfluorometry, and measurements of 86Rb+ uptake. Increasing osmolarity from 300 to 400 mosm/liter by addition of sucrose decreased cell volumes to 88.6% within 1 min; thereafter, cell volumes increased to 94.1% of control within 10 min, equivalent to a regulatory volume increase (RVI) by 44.5%. This RVI was paralleled by a decrease in cell input resistance and in specific cell membrane resistance to 88 and 60%, respectively. Ion substitution experiments (high K+, low Na+, low Cl-) revealed that these membrane effects are due to an increase in hepatocyte Na+ conductance. During RVI, ouabain-sensitive 86Rb+ uptake was augmented to 141% of control, and cell Na+ and cell K+ increased to 148 and 180%, respectively. The RVI, the increases in Na+ conductance and cell Na+, as well as the activation of Na+/K(+)-ATPase were completely blocked by 10(-5) mol/liter amiloride. At this concentration, amiloride had no effect on osmotically induced cell alkalinization via Na+/H+ exchange. When osmolarity was increased from 220 to 300 mosm/liter (by readdition of sucrose after a preperiod of 15 min in which the cells underwent a regulatory volume decrease, RVD) cell volumes initially decreased to 81.5%; thereafter cell volumes increased to 90.8% of control. This post-RVD-RVI of 55.0% is also mediated by an increase in Na+ conductance. We conclude that rat hepatocytes in confluent primary culture are capable of RVI as well as of post-RVD-RVI. In this system, hypertonic stress leads to a considerable increase in cell membrane Na+ conductance. In concert with conductive Na+ influx, cell K+ is then increased via activation of Na+/K(+)-ATPase. An additional role of Na+/H+ exchange in the volume regulation of rat hepatocytes remains to be defined.

The epithelial Na(+) channel (ENaC) is related to the hypertonicity-induced Na(+) conductance in rat hepatocytes.

The epithelial Na(+) channel (ENaC) is composed of the subunits alpha, beta, and gamma [Canessa et al., Nature 367 (1994) 463-467] and typically exhibits a high affinity to amiloride [Canessa et al., Nature 361 (1993) 467-470]. When expressed in Xenopus oocytes, conflicting results were reported concerning the osmo-sensitivity of the channel [Ji et al., Am. J. Physiol. 275 (1998) C1182-C1190; Hawayda and Subramanyam, J. Gen. Physiol. 112 (1998) 97-111; Rossier, J. Gen. Physiol. 112 (1998) 95-96]. Rat hepatocytes were the first system in which amiloride-sensitive sodium currents in response to hypertonic stress were reported [Wehner et al., J. Gen. Physiol. 105 (1995) 507-535; Wehner et al., Physiologist 40 (1997) A-4]. Moreover, all three ENaC subunits are expressed in these cells [Böhmer et al., Cell. Physiol. Biochem. 10 (2000) 187-194]. Here, we injected specific antisense oligonucleotides directed against alpha-rENaC into single rat hepatocytes in confluent primary culture and found an inhibition of hypertonicity-induced Na(+) currents by 70%. This is the first direct evidence for a role of the ENaC in cell volume regulation.

Naloxone-insensitive transport effects of loperamide in guinea-pig gallbladder epithelium.

The effects of the antidiarrheal drug, loperamide, on HCO3 and Na transport across guinea-pig gallbladder epithelium were investigated using Ussing-chamber methods. Under basal conditions, mucosal loperamide (10(-4) mol/l) moderately lowered both the absorptive (JHCO3ms) and the secretory HCO3 flux (JHCO3sm) (pH-stat method), most likely by changing paracellular HCO3 flow. Exposure to serosal prostaglandin E1 (10(-6) mol/l) abolished Na absorption and turned HCO3 secretion electrogenic. The associated short-circuit current (Isc) was inhibited by loperamide in a concentration-dependent manner; mucosal addition (threshold at 3 x 10(-6) mol/l) of the drug was more effective. Inhibition of Isc was related to a decrease in JHCO3sm, but exceeded the drop in JHCO3net. The effects on JHCO3sm and Isc were mimicked by [Met5]enkephalin. Naloxone (10(-6) mol/l) was unable to influence the effects of loperamide and [Met5]enkephalin on Isc. There were no pro-absorptive effects of loperamide on unidirectional Na fluxes. We conclude that antisecretory properties of loperamide are solely due to inhibition of electrogenic HCO3 secretion, an effect unrelated to opiate receptor binding.

Microbial Ecology of the Mango Phylloplane.

Bacteria, filamentous fungi, and yeasts were enumerated on the mango phylloplane by indirect leaf impression and washing- and dilution plating. The phylloplane microbial community was qualitatively and quantitatively related to leaf age, position in the tree canopy, seasonality, and chemical spraying. Filamentous fungi and yeasts were more abundant during winter and spring, whereas bacterial population densities increased during autumn. Community density and diversity increased progressively with leaf age. The western tree aspect sustained the least diverse bacterial, filamentous fungal, and yeast communities. Chemical sprays reduced bacterial, filamentous fungal, and yeast community diversities. Gram-positive bacteria (Bacillus spp. and coryneform spp.) exceeded gram-negative bacteria. The most common fungal genera isolated were Cladosporium and Alternaria. Yeasts prevalent in the mango phylloplane were of the genera Aureobasidium, Cryptococcus, and Sporobolomyces.

Mutagenicity of marijuana and Transkei tobacco smoke condensates in the Salmonella/microsome assay.

Extracts and smoke condensates of marijuana, Transkei home-grown tobacco and also commercial cigarette tobaccos were assayed for their mutagenic activity to Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538, both with and without metabolic activation. No mutagenic activity was detected in dichloromethane extracts of marijuana and tobacco per se, but all the smoke condensates exhibited mutagenicity with metabolic activation. The only strain not mutated by any of the pyrolyzates was TA1535. Transkei tobacco pyrolyzate proved to be the most mutagenic, followed by marijuana, pipe and cigarette tobacco. Mutagenicity was positively associated with the nitrogen content of the various products. The potent mutagenic action of marijuana smoke condensate, coupled with a condensate yield of more than 50% higher than that of cigarette and pipe tobacco, indicates a high carcinogenic risk associated with marijuana smoking.

Mutagenicity to Salmonella typhimurium of some Aspergillus and Penicillium mycotoxins.

17 mycotoxins produced by various Aspergillus and Penicillium species were screened for their mutagenic activity to Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, both with and without metabolic activation. Austdiol, austocystins A and D, kojic acid and viridicatumtoxin were found to be mutagenic after metabolic activation, while austdiol was also mutagenic per se. Aflatoxin B1, sterigmatocystin and versicolorin A, which were used as positive controls were also mutagenic. No mutagenic activity was evident in the case of citrinin, cyclopiazonic acid, fumitremorgen B, griseofulvin, luteoskyrin, O-methylsterigmatocystin, mycophenolic acid, ochratoxin A, patulin, penicillic acid, secalonic acid D and TR2-toxin. A good relationship was found between the mutagenic activity, or lack of it, of most of the mycotoxins with existing data on carcinogenicity. Inadequate information on the carcinogenicity of austdiol, austocystins A and D, kojic acid and viridicatumtoxin precluded correlations with mutagenicity to S. typhimurium. The relationship between chemical structure and mutagenicity of the mycotoxins is discussed.

Characterization of the Crater Disease Strain of Rhizoctonia solani.

ABSTRACT Crater disease (CD) of wheat is caused by a Rhizoctonia solani strain of ambiguous phylogeny. Anastomosis reactions confirmed placement of CD-causing R. solani in anastomosis group (AG) 6, with results indicating a closer affinity to AG-6 GV than to AG-6 HG. Cultures of CD isolates were initially white to cream, turning a yellowish light brown after 10 days. Concentric rings of dark and light mycelium were evident from an early stage. Mycelium generally was appressed to the agar surface, with sparse aerial growth. A few light-colored, irregularly shaped sclerotia could be discerned after 2 weeks. The mean hyphal diameter of CD-causing R. solani was 7.46 mum (ranging from 5.0 to 10.0 mum), and cells contained a mean number of four (ranging from two to eight) nuclei, compared to a mean hyphal diameter of 8.58 and 8.42 mum and a mean nuclear number of six and four for AG-6 HG and AG-6 GV, respectively. The CD isolates had a slower growth rate (15.3 mm/day) than AG-6 HG (29.1 mm/day) and AG-6 GV (22.6 mm/day) but, like AG-6, were thiamine prototrophic. Conspicuous nodulose swellings were produced by CD-causing R. solani on roots of wheat, and infection resulted in retarded shoot growth. Smaller nodules were evident on bean and soybean roots. Fingerprint patterns generated for the various isolates with four enzymes, HpaII, Sau3AI, TaqI, and CfoI, showed the presence of a unique 610-bp fragment in the pathogen. It is proposed that CD-causing R. solani isolates represent a distinct intersterility group within AG-6 that is more related to subgroup GV than to subgroup HG.

Delimitation of the time of death by immunohistochemical detection of glucagon in pancreatic alpha-cells.

To improve the possibilities of delimitating the time of death after longer laytime it was examined if this is possible by immunohistochemical glucagon detection. The results show that in our examination material the pancreatic alpha-cells of up to 6-day-old corpses produce a positive immunoreaction towards glucagon in all cases whereas none of the corpses older than 14 days show such a reaction. This means that in the case of a negative immunoreaction the time of death can be assumed to lie more than 7 days before the autopsy. The fact that a negative immunoreaction occurs consistently after 14 days leads to the conclusion that when glucagon has been stained in a specimen, the death of the respective person must lie a maximum of 13 days earlier, whereby under markedly different conditions to the ones of the cases here examined, a negative immunoreaction could happen earlier and a positive immunoreaction even later.

Delimitation of the time of death by immunohistochemical detection of calcitonin.

To improve the possibilities of delimitating the time of death after longer laytime it was examined if this is possible by immunohistochemical detection of calcitonin. The results show that in our examination material the c-cells of the thyroid glands of up to 4-day-old corpses produce a positive immunoreaction towards calcitonin in all cases whereas none of the corpses older than 13 days show such a reaction. This means that in the case of a negative immunoreaction the time of death can be assumed to lie >4 days before the autopsy. The fact that a negative immunoreaction occurred consistently after 13 days leads to the conclusion that when calcitonin has been stained in a specimen, the death of the respective person must lie a maximum of 12 days earlier, whereby these time-limits may change in considerably different surrounding conditions.

Percentile charts to determine the duration of child abuse by chronic malnutrition.

Longstanding quantitative or qualitative under-supply of nutrition leads to weight loss and, in children, to stagnation of growth and thus to stunted growth. A comparison of the expected growth, according to percentile growth curves, with the actual body size, gives an indication as to the period of time in which malnutrition took place. The moment in which the growth curve bends off and leaves the norm is to be interpreted as the earliest begin, the moment in which the attained growth would have been achieved as the latest begin of the nutritional impairment.

Delimitation of the time of death by immunohistochemical detection of thyroglobulin.

To improve the possibilities to delimit the time of death after longer laytime it was examined if this is possible by immunohistochemical detection of thyroglobulin. The results show that in our examination material the colloid and the follicular cells of the thyroid glands of up to 5-day-old corpses produce a positive immunoreaction towards thyroglobulin in all cases whereas none of the corpses older than 13 days show such a reaction. This means that in case of a negative immunoreaction the time of death can be assumed to lie more than 6 days before the autopsy. The fact that a negative immunoreaction occurs consistently after 13 days leads to the conclusion that when thyroglobulin has been stained in a specimen, the death of the respective person must lie a maximum of 12 days earlier, whereby these time-limits may change in considerably different surrounding conditions.

Insulin- or morphine-injection? Immunohistochemical contribution to the elucidation of a case.

Two autopsy cases of an elderly couple who died on the same day will be used to underline the importance of immunohistochemistry of forensic practice. At first unexplainable injection marks on the upper arms of the corpses and the possibility of a closely related physician injecting insulin and certifying a natural death made it important, considering suspect insulin concentrations in the blood, to exclude insulin injections in these marks. Further, the statement that morphine had been injected for the analgesia of tumour pains, was reinforced by immunohistochemistry.

Streifenlichttopometrie (SLT): a new method for the three-dimensional photorealistic forensic documentation in colour.

By means of the new method of Streifenlichttopometrie (SLT) it is possible to record the complete body surface of casualties in a practically photorealistic fashion, i.e. three-dimensionally and in colour. In comparison with the classic method of Photogrammetry Streifenlichttopometrie (SLT) is remarkably faster (10,000 points/s instead of 1 point/s) and in addition the colour of every point measured upon the corpse's surface is instantly recorded. Taking into consideration the resolution required and the qualities of the camera system the body surface is recorded in 'patches', i.e., areas of a defined extension (in the present case 500 mmx500 mmx200 mm) which are marked with a body fixed reference frame to grant the exact matching of the data after the recording process. Length, perimeter, square and volume of the body segments and injuries can be determined. Furthermore the natural colour of the wounds can be used for an immediate classification according to the intensity of the impact forces. In addition the 3-D coordinates of the body surface including the wounds can be transferred into an animated computer simulation for the reconstruction of the traumatic events.

Immunohistochemical detection of methadone in the human brain.

To develop a method of detecting methadone in the human brain by immunohistochemistry, brain tissue of frontal cortex, cerebellum, hippocampus, basal ganglions and brain stem from victims of a lethal methadone overdose was examined. The staining was performed with a monoclonal anti-methadone antibody from the mouse, originally developed for immunochemichal purposes (ELISA). With the help of the DAKO((R)) Catalyzed Signal Amplification (CSA) System, a specific positive immunoreaction was obtained in the neurons of the frontal cortex and hippocampus, as compared with specimen from deaths without exposition to methadone. Thus, along with metamphetamine, phenobarbital, morphine and insulin, immunohistochemical detection is also possible for methadone and the intake of this medicament can now be proven morphologically.

Delimitation of the time of death by immunohistochemical detection of insulin in pancreatic beta-cells.

To improve the possibilities to delimitate the time of death after longer laytime, it was examined if this is possible by immunohistochemical insulin detection. The results show that in our examination material, the pancreatic beta-cells of up to 12-day-old corpses produce a positive immunoreaction towards insulin in all cases, whereas none of the corpses older than 30 days show such a reaction. This means that in case of a negative immunoreaction, the time of death can be assumed to lie more than 12 days before the autopsy. The fact that a negative immunoreaction occurs consistently after 30 days leads to the conclusion that when insulin has been stained in a specimen, the death of the respective person must lie a maximum of 29 days earlier, whereby these time-limits may change in considerably different surrounding conditions.