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The cytotoxins from Clostridium difficile (toxin B) and Clostridium sordellii (toxin L) induce rounding of cultured cells. The cellular effects induced by these two cytotoxins are clearly distinct, suggesting that both toxins use a similar, but not identical mechanism for cell intoxication. We have employed the technique of two-dimensional PAGE for the separation of 32P-labelled cell lysates of McCoy cultured cells to investigate changes in the phosphorylation status of cellular proteins after treatment with toxin B and with toxin L. The two-dimensional electrophoresis patterns suggest the implication of an 80,000 mol. wt cellular protein (named pp80c) in the cytopathic action of the cytotoxin from C. sordellii. This protein shows immunoreactivity with non-muscle caldesmon. Toxin B, however, does not affect the phosphorylation of pp80c, but alters the phosphorylation of another cellular protein, pp77, indicating another mechanism for cell intoxication. In addition, our experiments suggest that the mechanism of action of okadaic acid, a phosphatase inhibitor which causes cell rounding similar to that induced by C. sordellii, and these two cytotoxins are different.

Stool samples (355 from 350 patients) were examined in a new commercial assay, the ToxA test, for the rapid diagnosis of Clostridium difficile-associated diarrhoea. The results were compared with direct assay of cytotoxin in McCoy cell tissue culture, and detection of toxigenic C. difficile by culture and cytotoxin testing of isolates. Discordant results were resolved by consultation of clinical records. Test sensitivities were 84.6% for the ToxA test, 78.5% for the direct cytotoxicity assay and 95.4% for culture. The specificity was 100% for all the tests. The ToxA test is a rapid and reliable alternative for the detection of toxigenic C. difficile where tissue culture facilities are unavailable.

Exposure of McCoy cultured cells to Clostridium difficile cytotoxin B or okadaic acid (OA), a potent phosphatase inhibitor, results in similar morphological changes. Using two-dimensional gel electrophoresis, we have detected a protein of approximately 77 kDa, with a pI of 4.5 (termed pp77) which is hyperphosphorylated in both cases. The level of phosphorylation of pp77 is increased by 293% and 35% after treatment with C. difficile cytotoxin B or OA, respectively. This protein was identified by microsequencing as calnexin, a protein which exhibits the characteristics of an endoplasmic reticulum (ER) chaperone.

A 33 basepair oligonucleotide probe, designed from the sequence of the Clostridium difficile toxin B gene, was evaluated for its ability to detect toxigenic Clostridium difficile directly in stool samples, without culture or DNA isolation. Two different labelling techniques were investigated: radiolabelling and digoxigenin-labelling. One hundred ninety-six stools were tested, with a good correlation (96%) obtained between the oligonucleotide probe and the gold standard, the cytotoxicity tissue culture assay. The sensitivity and specificity were 83% and 100%, respectively. In parallel, a new commercially available enzyme immunoassay for the detection of Clostridium difficile toxin A in stool specimens was investigated. In 162 samples tested, a sensitivity of 80% and a specificity of 98% were obtained.

Fluorescence resonance energy transfer (FRET) was used to study hybrid formation and dissociation after microinjection of oligonucleotides (ODNs) into living cells. A 28-mer phosphodiester ODN (+PD) was synthesized and labeled with a 3' rhodamine (+PD-R). The complementary, antisense 5'-fluorescein labeled phosphorothioate ODN (-PT-F) was specifically quenched by addition of the +PD-R. In solution, the -PT-F/+PD-R hybrid had a denaturation temperature of 65 +/- 3 degrees C detected by both absorbance and FRET. Hybridization between the ODNs occurred within 1 minute at 17 microM and was not appreciably affected by the presence of non-specific DNA. The pre-formed hybrid slowly dissociated (T1/2 approximately 3 h) in the presence of a 300-fold excess of the unlabeled complementary ODN and could be degraded by DNAse I. Upon microinjection into the cytoplasm of cells, pre-formed fluorescent hybrids dissociated with a half-time of 15 minutes, which is attributed to the degradation of the phosphodiester. Formation of the hybrid from sequentially injected ODNs was detected by FRET transiently in the cytoplasm and later in the cell nucleus, where nearly all injected ODNs accumulate. This suggests that antisense ODNs can hybridize to an intracellular target, of exogenous origin in these studies, in both the cytoplasm and the nucleus.

Two case reports of critically ill neonates, one with pneumomediastinum, the other with pneumopericardium, illustrate how the inability to view the heart through the subxiphoid echocardiographic window occurs only in pneumopericardium. This echocardiographic nonfinding should alert the echocardiographer and neonatal team of impending pericardial tamponade.

A 32-yr-old female presented to Sports Clinic with bilateral ankle pain and swelling following the initiation of an aerobic dance program 3 months earlier. Physical exam revealed bilateral ankle edema and the recent appearance of discrete tender nodules on her anterior tibias, consistent with erythema nodosum (EN). As part of her evaluation, chest radiographs were performed and demonstrated bilateral hilar adenopathy. This case discusses the evaluation of the patient with joint involvement, EN, and hilar adenopathy. The increased accessibility of sports medicine clinics makes it incumbent on the physician to be familiar with a wide range of differential diagnoses.

This article was written in May 1992, after Magic Johnson announced he had tested positive for the human immunodeficiency virus that causes AIDS and was retiring from the Los Angeles Lakers. Since then, Johnson played in the 1992 Summer Olympic Games and, after a brief return to the Lakers, permanently retired from the NBA.

We describe the use of a new specific synthetic oligonucleotide probe, deduced from the sequence of the gene for Clostridium difficile toxin B, to identify toxigenic strains of C. difficile. This probe does not hybridize to the DNA of non-toxigenic strains of C. difficile nor to DNA isolated from different Clostridium species, including C. sordellii. None of the enteric pathogenic bacteria tested were seen to hybridize with the probe. A preliminary study of direct probing of faecal specimens indicates a potential for the use of this DNA probe in the clinical laboratory for the rapid identification of toxigenic strains of C. difficile.

This article presents an overview of some of the more commonly encountered disorders of the gastrointestinal tract that are seen in athletes. As with other medical conditions, the clinician can often be blinded by an athlete's physical activity and overlook underlying disease. Fortunately, most of the maladies described here are amenable to medical treatment, and with proper diagnosis and management the athlete should be able to achieve his or her maximal athletic performance. The study of the gastrointestinal tract and exercise is still in its early stages and was prompted by the need to explain clinical observations. Although some questions have been answered, the positive and negative effects of physical activity on the gastrointestinal tract have many avenues of research left to explore.

We compared the immunological properties and cytotoxic effects of Clostridium difficile toxin B and Clostridium sordellii toxin L. These two cytotoxins are immunologically related in that the cytotoxic effect of either toxin can be neutralized by the polyclonal antiserum prepared against either cytotoxin. On the other hand, polyclonal antiserum prepared against Clostridium difficile enterotoxin A did not cross-react with the cytotoxins B and L when tested by cytotoxic neutralization test nor by double immunodiffusion assay. However, despite this immunological relationship between toxins B and L, the morphological modifications observed in MacCoy cells induced by treatment with these cytotoxins are clearly distinct. We describe the first quantitative analysis of specific cellular parameters which illustrates the morphological differences induced by these cytotoxins. Moreover, immunocytochemical experiments show that, whereas disruption of microfilaments is observed with toxin B- and L-treatments, alterations of F-actin network are different in the cells treated with toxin B or L. The observation that the cellular modifications induced by toxin B- and toxin L-treatment differ suggests that the molecular mechanisms involved in the respective cytotoxicities are also likely to be different.

Mercury-resistance genes were detected byin situ hybridization using new synthetic oligonucleotide probes specific formerA andmerB genes according to the published sequences of the corresponding enzymes. These DNA probes were used for the detection of specific mercury-resistant microorganisms isolated from the Rhine River which had been polluted 3 years previously in 1986. Mercuric reductase and organomercurial lyase genes persist in the bacterial genome even after the disappearance of the pollutant but are absent in axenic amoebae. A total of 49 bacterial isolates showed DNA homologies with the(32)P-labelled DNA probes and 15 free-living amoebae were selected due to their harboured symbiotic mercury-resistant bacteria.

This study demonstrates that low-density metabolically replete HbSS erythrocytes suspended in heat-inactivated autologous plasma and subjected to 15 hr of cyclical deoxygenation (under nitrogen) bind significantly increased quantities of autologous IgG as compared with oxygenated paired samples. IgG binding to the erythrocyte surface was quantified by a nonequilibrium 125-iodinated protein A binding assay and by flow cytometry. Sickle cells deoxygenated 15 hr (37 degrees C) in the presence of 2 mM calcium bound 2.2 +/- 0.2 (mean +/- SD)-fold more IgG (p less than 0.01) than oxygenated paired samples. Sickle erythrocytes deoxygenated in 0.4 mM EDTA bound 1.7 +/- 0.3 (mean +/- SD)-fold more autologous IgG than oxygenated controls (p less than 0.05). Indirect immunofluorescence assays also demonstrated that the relative levels of autologous IgG bound to sickle cells after 15 hr cyclical deoxygenation in the presence or absence of calcium was increased as compared with IgG binding by oxygenated paired samples. After 3 hr of cyclical deoxygenation in the presence of 2 mM calcium sickle erythrocytes exhibited a 40-60% increase in IgG binding, as compared with 10-20% increased IgG binding by paired samples treated in EDTA. These findings demonstrate that repeated morphologic sickling will increase the IgG binding capacity of low-density sickle cells, and suggest that sickling-associated alterations of the cell surface will produce new binding sites recognized by autologous IgG. These studies also show that the sickling-induced increase in IgG binding may be slightly enhanced by the presence of extracellular calcium.

Cows (890) in 15 US herds were assigned randomly in equal numbers to control or bST injections (500 mg in a prolonged-release form every 14 d for 12 wk) within three stages of lactation (57 to 100, 101 to 140, and 141 to 189 d postpartum) and two parity groups (primiparous and multiparous). Yield and milk composition were monitored 1 d/wk for 16 wk including 2 wk pretreatment and 2 wk posttreatment. Increases in milk and FCM due to bST injections were less at 57 to 100 d than at 101 to 189 d postpartum (milk 3.6 vs. 5.5; FCM 3.9 vs. 6.1 kg/d per cow), and increases in milk and FCM were more for multiparous than for primiparous cows (milk 5.5 vs. 4.2; FCM 6.0 vs. 4.7 kg/d cow). Temporarily, concentration of milk fat increased and protein decreased; later, concentrations for control and injected cows were similar. Postinjection milk fat concentration decreased, but milk protein concentration increased temporarily. The net increase in milk (and FCM) varied significantly among herds from 2.9 to 7.6 kg/d per cow (mean, 4.9 kg). Responses in FCM were similar over a wide range of pretreatment yields. A great variety of feed ingredients were fed as total mixed rations, and nutrient concentrations varied greatly. The SCC were similar before, during, and after treatment, but increase in FCM of injected cows exhibited a negative correlation with pretreatment SCC. Changes in body condition score of sometribove-injected cows varied among herds (.25 to -.45) and averaged -.02 compared with .07 for controls. There was no pattern in incidence of mastitis during sometribove injections.

The intracellular transport and fate of nucleic acids is poorly understood. To study this process, we injected fluorescent oligodeoxyribonucleotides (oligos) into the cytoplasm of CV-1 epithelial cells and primary human fibroblasts. Rapid nuclear accumulation was found with the phosphodiester (PD), phosphorothioate (PT), and methylphosphonate (MP) forms of a 28-mer oligo complimentary to the rev mRNA of the human immunodeficiency virus type 1. Migration of the oligos in the cytoplasm was slower than diffusion of a coinjected dextran, but the oligos freely diffused into the nucleus. Nuclear incorporation was temperature but not energy dependent. The intranuclear distribution of the oligos was influenced by the chemistry of internucleoside linkages. The PD oligos and, to a lesser extent, the PT oligos colocalized with small nuclear ribonucleoproteins (snRNPs), whereas the MP oligos colocalized with concentrated regions of genomic DNA. These data have important implications for our understanding of the transport and accumulation of exogenous nucleic acids in mammalian nuclei, and the assay described could potentially be used for testing the efficacy of oligos designed as therapeutic agents.

We have developed a rapid method for the purification of proteins, combining titration curve analysis with a two-step column chromatographic procedure. We have used this approach to purify the cytotoxin (L toxin) from Clostridium sordellii. We have also determined the amino acid composition of this cytotoxin. This toxin has a pI value of 4.20 and an Mr of 260,000, reduction of which results in a band of Mr 43,000 on sodium dodecyl sulphate polyacrylamide gel electrophoresis. Since both the proteins of Mr 260,000 and 43,000 are recognized by the polyclonal anti-C. sordellii L toxin, which neutralizes the L toxin cytotoxicity, we propose a hexameric structure for the protein of Mr 260,000, each subunit being Mr 43,000.

Excessive binding of possible autoantibodies to discrete subpopulations of circulating sickle erythrocytes has been documented. However, the mechanism by which putative autoantibody binding sites selectively develop on sickle cells is unknown. In the present study, autologous IgG binding has been quantified for low density sickle erythrocytes subjected to prolonged morphologic sickling under nitrogen in the absence of plasma (24 hours, 37 degrees C), and to reoxygenation with subsequent incubations in varying dilutions of autologous plasma. Cell-bound IgG was measured using a nonequilibrium 125-iodinated protein-A-binding assay. Binding isotherms show that IgG binding was both concentration dependent and saturable. Sickle cells pretreated by deoxygenation exhibited approximately 2-fold increased saturation binding of autologous IgG as compared with oxygenated paired samples, suggesting that new autoantibody binding sites may have developed during prolonged sickling. Autologous IgG binding to sickle cells pretreated by deoxygenation was also inhibited 2-fold more by limiting quantities of deoxygenated autologous cells, as compared with inhibition by oxygenated sickle erythrocytes used for serum absorption. These findings indicate that the sickling-associated increase in IgG binding may represent an increase in specific autoantibody binding sites, and suggests that autoantibody binding sites are produced by permanent sickling-associated remodeling of the red cell surface.

This study reports that sickling-induced increased autoantibody binding can be demonstrated in varying degrees for deoxygenated S/beta-thalassemic (2-fold) and hemoglobin-SC (1.2-fold) erythrocytes as compared with oxygenated paired samples. In contrast, HbAS erythrocytes deoxygenated in autologous plasma exhibited less than 2% morphologic sickling and no increased IgG binding as compared with control samples. Sickling in the presence or absence of plasma increased the IgG binding capacity of S/beta-thalassemic erythrocytes, comparable to previous findings for HbSS erythrocytes, while increased IgG binding to HbSC erythrocytes was detected only after deoxygenation in plasma. It is concluded that specific IgG binding to deoxygenated S/beta-thalassemic RBCs results from subtle permanent sickling-induced alterations of the membrane surface, while IgG binding to HbSC erythrocytes sickled in plasma results from transitory membrane changes. These findings suggest that sickling in vivo will produce cumulative autoantibody binding to S/beta-thalassemic erythrocytes, a process which could lead to immune-mediated erythrocyte destruction. In contrast, comparatively small fractions of the autoantibody bound to HbSC erythrocytes in vivo would result from sickling-induced membrane alterations. These studies indicate that sickling-associated autoantibody binding in vivo will not occur for sickle cell trait (HbAS) erythrocytes protected by plasma.

The object of this study was to determine the importance of domestic cats in the epidemiology of human giardiasis. Of six laboratory-reared cats inoculated with cultured Giardia lamblia trophozoites from humans, only one showed the presence of cysts in the feces, and cysts were found on only 1 of the 80 days of observation. In a second experiment, eight cats were inoculated with G. lamblia cysts isolated from a human being. Over an 8-week period of observation, two of eight cats were found to have passed cysts in their feces, one on only one day and the other on 2 days. Postmortem examination of all of the cats found to be passing G. lamblia cysts at some time during the experiments did not reveal any small-intestinal trophozoites. These results suggest that domestic cats are relatively insusceptible to G. lamblia from humans and, consequently, that cats probably are not significant reservoir hosts of Giardia spp. infective for human beings. Moreover, it appears that the Giardia spp. which parasitize cats are distinct from those of human beings.

A group of experts from the University of California, Los Angeles, met to discuss a case from another institution. This case conference is part of a series featuring a variety of sports medicine topics.

Methyl group turnover rates for specific methyl-accepting membrane proteins in intact irreversibly sickled cells (ISCs) have been determined. The turnover of methyl groups on all methyl acceptor membrane proteins carboxylmethylated in ISCs is not concerted but proceeds in an ordered sequence which is markedly different from that exhibited by unfractionated normal erythrocytes (AA). In ISCs methyl group turnover based on initial demethylation rate constants is most rapid for membrane polypeptides migrating in sodium dodecyl sulfate at 30,000-39,000 Da, 40,000-55,000 Da, polypeptides comigrating with cytoskeletal component band 4.1, and band 4.5. In contrast, initial methyl group turnover rates obtained after less than 20% of the total methyl groups are turned over in (AA) cells show most rapid demethylation rates for membrane polypeptides migrating at 40,000-55,000 Da, polypeptides comigrating with band 4.5, cytoskeletal components bands 2.1 and 4.1. Results also show significant differences between ISCs and most dense fractions from normal (AA) and nonsickle hemolytic anemias in the demethylation of cytoskeletal proteins, bands 2.1 and 4.1. These findings indicate qualitative differences in accessibility of methyl acceptor substrates to the methylating-demethylating enzyme activity in the cytosol of irreversibly sickled cells compared to discocytic control erythrocytes.

We report here that the sequence ofP. vulgaris mitochondrial and cytoplasmic tRNA(Leu) (NAA) are identical except for a post-transcriptional modification. There is an unidentified modification at the "wobble" position which, from the sequence of the nuclear tRNA(Leu) gene, we identify as a derivative of C. We also show that thisP. vulgaris nuclear gene is functional by demonstrating its transcription in anin vivo eukaryotic transcription system.

The nucleotide sequence of the cytoplasmic 5S ribosomal RNA from Scenedesmusobliquus has been determined using post-labelling techniques. The sequence is closely related to the 5S RNA sequence of Chlorella and contains all the areas of invariant sequence which appear to be conserved in plant cytoplasmic 5S RNA species.

A genomic bank of nuclear DNA (nDNA) from the higher plant Phaseolus vulgaris, constructed using the lambda EMBL-4 vector, has been screened for the presence of tRNA genes. One of the many positive recombinants was found to hybridise several times stronger than the other positives, and has been shown to contain several tRNA genes. We report the structure of two nuclear tRNA genes for tRNA(Pro), namely tRNA(Pro)(UGG) and tRNA(Pro)(AGG), and that of a 'pseudogene' for tRNA(Pro). This 'pseudogene', despite showing 95% homology with the other tRNA(Pro) species presented here, has several features which are likely to affect its transcription or its functioning as a tRNA.

Increases in brain stem blood flow (BBF) during hypoxia may decrease tissue PCO2/[H+], causing minute ventilation (VE) to decrease. To determine whether an increase in BBF, isolated from changes in arterial PO2 and PCO2, can affect respiration, we obstructed the thoracic aorta with a balloon in 31 intact and 24 peripherally chemobarodenervated, anesthetized, spontaneously breathing newborn piglets. Continuous measurements of cardiorespiratory variables were made before and during 2 min of aortic obstruction. Radiolabeled microspheres were used to measure BBF before and approximately 30 s after balloon inflation in eight intact and five denervated animals. After balloon inflation, there was a rapid increase in mean blood pressure in both the intact and denervated animals, followed within 10 s by a decrease in tidal volume and VE. In the intact animals, the decrease in VE after acute hypertension can be ascribed to a baroreceptor-mediated reflex. After peripheral chemobarodenervation, however, acute hypertension continued to produce a decrease in VE, which cannot be explained by baroreceptor stimulation. In these denervated animals, aortic balloon inflation was associated with an increase in BBF (13.1 +/- 2.7%; P less than 0.05). We speculate that the increase in BBF during hypoxia may contribute to the decrease in ventilation observed after carotid body denervation.

Adrenal glucocorticoids exert a permissive action on the glycogen phosphorylase cascade. Epinephrine activation of muscle phosphorylase and phosphorylase b kinase is depressed in adrenalectomized rats. Phosphorylase phosphatase activity is increased by steroid lack, and normal epinephrine inhibition of the enzyme does not occur. Phosphorylase b kinase phosphatase activity is also increased; epinephrine, however, does not inhibit activity in muscle from normal or adrenalectomized rats. Protein phosphatase inhibitor activity is depressed in boiled dialyzed preparations made from adrenalectomized rat muscle. Cortisol resplacement therapy restores protein phosphatase inhibitor activity, decreases increased protein phosphatase activity, and restores normal epinephrine-induced activation of phosphorylase b kinase and phosphorylase and epinephrine inhibition of phosphorylase phosphatase.

Two dose rates and 2 management systems for the synchronization of estrus in cattle, using prostaglandin F 2 alpha (PGF2 alpha), were compared within 3 dairy and 3 beef herds. There was no statistical difference in responses between herds or breeds. There was no statistical difference between the estrous response produced by the 12.5-mg dose rate (53.6%) and that produced by the 25-mg dose rate (56.7%, P = 0.5). The 10-day, 1-injection management system produced a 21.7% better estrous response than that produced by the 2-injection system (64.5% vs 42.8%, respectively; P = 0.0001). Most (79.5%) of the difference in estrous response between management systems was accounted for by cows not responding to the 2nd injection of the 2-injection system. The interval from injection to estrus after 1 PGF2 alpha injection was shorter than after 2 PGF2 alpha injections (63.6 +/- 25.2 vs 70.3 +/- 23.8 hours, respectively). The percentage of all cows in the artificial insemination program that were in estrus 56 to 80 hours after injection was 27.4%.

The nucleotide sequences of two species of tyrosine accepting tRNA from the eukaryotic green alga Scenedesmus obliquus have been determined. The sequence of the cytoplasmic tRNATyr is: (sequence in text) This is the first chloroplast tRNATyr species to be sequenced.

The nucleotide sequence of a cytoplasmic tRNAPhe from the eukaryotic green alga Scenedesmus obliquus was determined as: pG-G-C-U-U-G-A-U-A-m2G-C-U-C-A-G-C-D-Gm-G-G-A-G-A-G-C-m22G-p si-psi-A-G-A-Cm-U-G - A-A-m1G-A-psi-C-U-A-C-A-G-m7G-N-m5C-C-C-C-A-G-T-psi-C-G-m1A-U-m5C-Cm-U-G -G-G-U- C A-G-G-C-C-A-C-C-A-OH. The structure has some notable features. Unlike other tRNAPhe species from plant sources, it has an unmodified G as the first residue of the anticodon and m1G rather than a Y derivative as the residue following the anticodon. The sequence m5C(60)-Cm(61) is unique to this tRNA. The sequence of S. obliquus tRNAPhe shows close homology with S. obliquus tRNATyr.

The transfer of detergent solubilized and purified gamma-glutamyl transpeptidase (gamma-GTase), of hog kidney cortex, from proteoliposomes into human erythrocyte ghost membranes has been studied. The transfer of gamma-glutamyl transpeptidase was observed upon incubation of gamma-GTase incorporated dipalmitoylphosphatidylcholine vesicles with erythrocyte ghost membranes at 37 degrees C for 12 h. The extent of transfer was dependent upon the fluidity of donor proteoliposomes, being more when dipalmitoylphosphatidylcholine proteoliposomes were used compared to dimyristoylphosphatidylcholine, and intermediate values were observed when binary mixtures of DMPC and DPPC were used. Moreover, the transfer of gamma-GTase was facilitated when rigid basic phospholipid proteoliposomes were used as donor. The transfer of gamma-GTase has been observed to be associated with the removal of intrinsic membrane proteins and lipids from erythrocytes, mainly acetylcholinesterase, sphingomyelin, and cholesterol. An enhancement in the fluorescence due to resonance energy transfer was observed when ghost membranes containing fluorescent donor probe were incubated with proteoliposomes containing fluorescent acceptor probe, indicating that fusion but not adsorption of vesicles occurs during the transfer process. However, the inability of entrapped [14C]-sucrose delivery from proteoliposomes into ghost membrane vesicle suggest that fusion per se is not primarily involved in the transfer process. It appears that the transfer of gamma-glutamyl transpeptidase occurs by a collisional transfer process resulting in intermembrane protein transfer. The gamma-glutamyl transpeptidase implanted ghost membranes exhibited the uptake of L-glutamate which was inhibited by serine-borate, an inhibitor of transpeptidase activity. In addition, the uptake of L-glutamate was inhibited by the dipeptide gamma-glutamyl-L-glutamate, thus supporting the proposed role of gamma-glutamyl transpeptidase in the uptake of amino acids in biological membranes.