Non-invasive detection of malignancy by identification of unusual CD44 gene activity in exfoliated cancer cells.

OBJECTIVE--To investigate non-invasive detection of cancer by testing for unusual CD44 gene activity in a clinical sample as an indicator of exfoliated tumour cells. DESIGN--Case-control study. SUBJECTS--44 unselected, consecutive patients with bladder cancer and 46 people with no evidence of neoplasia. MAIN OUTCOME MEASURE--Presence or absence of large CD44 gene products containing exon 6 derivatives in urine samples. RESULTS--Novel abnormalities in the pattern of expression of this gene, seen specifically in tumour tissue, led to cloning of a newly recognised coding region in it (exon 6). This was tested as a probe for detection of exfoliated malignant cells in naturally voided urine. CD44 gene products extracted from the urine and amplified with polymerase chain reaction contained predicted electrophoretic band of 735 base pairs in 40 of the 44 patients with bladder cancer (sensitivity 91%). Products from 38 of the 46 people with no evidence of neoplasia showed no such band (specificity 83%). CONCLUSIONS--Unusual activity of the CD44 locus in neoplasia and malignancy is confirmed, and techniques for the analysis of such activity can enable non-invasive investigation of patients for primary or recurrent bladder cancer or for other tumours that shed neoplastic cells into body fluids.     

Diflubenzuron affects gamma-thioGTP stimulated Ca transport in vitro in intracellular vesicles from the integument of the newly molted american cockroach, Periplanet americana L.

To study the mechanism of action of diflubenzuron (DFB) and other benzoylphenylureas, we have initially hypothesized that their action may be related to exocytosis: to test the hypothesis, we obtained an intracellular vesicle preparation from the homogenate of integument of newly molted American cockroachs (Periplaneta americana L.) in 10 mM MES buffer containing 250 mM sucrose (isotonic) and 2.5 mM MgSO₄, at pH 6.6. By studying DFB's effect on various ion transporting activities, we demonstrated that calcium uptake in this intracellular particulate preparation was significantly inhibited by DFB at low concentrations (e.g., 10⁻⁸ M). Such an inhibitory effect of DFB on Ca²⁺ uptake was eliminated by the addition of ionophores or membrane disruptors, as well as the sonication of vesicle preparation. On the other hand, oligomycin, protein phosphorylation modulators, Na⁺, and Li⁺ did not affect the calcium uptake. Among ionophores, agents disrupting H⁺ gradients (e.g. FCCP and NEM) totally eliminated ⁴⁵Ca uptaking activity by vesicles as well as the inhibitory effect of DFB. Among calcium ion modulators, calmodulin inhibitors such as calmidazolium and trifluoperazine decreased the Ca²⁺-uptake, whereas membrane calcium channel blocker, verapamil, did not. ATP and γ-S-GTP stimulated Ca²⁺ uptake. However, the former increased only the DFB insensitive portion and the latter largely the DFB sensitive part of Ca²⁺. Together these data support the hypothesis that the action site of DFB in this preparation is the GTP-dependent Ca²⁺ transport process which is coupled to vacuolar type intracellular vesicles in the integument cells.     

Reexamination of tetrodotoxin production by bacteria.

Vibrio alginolyticus has been reported as a good producer of tetrodotoxin (TTX), but the toxin extracted from this bacterium did not react to the monoclonal antibody against TTX. Surprisingly, chromatographic analyses detected high TTX peaks for polypeptone and yeast extracts used as medium materials, which were, as expected, all negative by the mouse bioassay. These results may require us to revise the bacterial production of TTX.     

Application of solvent extraction to ethanol fermentation

Summary The basic problems of applying solvent extraction to ethanol fermentation were investigated. The selection of solvents was based on the selectivity ratio, which was expressed as the ratio of the ethanol distribution coefficient to the water distribution coefficient. Solvents with high selectivity ratios of more than 50 were found mainly among the alcohols and esters. However, most of these solvents were toxic to ethanol-producing microorganisms. We tried to make a barrier to solvent molecules beneath the surface of gel beads immobilizing the cells as a protection against solvent toxicity. Porapack Q was found to be an effective barrier, and the ethanol production rate of immobilized cells protected with Porapack Q did not change event after the production of eight batches in medium saturated with sec-octanol, which was the most toxic solvent used in our experiments.     

Gluconeogenesis predominates in periportal regions of the liver lobule

Rates of gluconeogenesis from lactate were calculated in periportal and pericentral regions of the liver lobule in perfused rat livers from increases in O2 uptake due to lactate. When lactate (0.1-2.0 mM) was infused into livers from fasted rats perfused in either anterograde or the retrograde direction, a good correlation (r = 0.97) between rates of glucose production and extra O2 uptake by the liver was observed as expected. Rates of oxygen uptake were determined subsequently in periportal and pericentral regions of the liver lobule by placing miniature oxygen electrodes on the liver surface and measuring the local change in oxygen concentration when the flow was stopped. Basal rates of oxygen uptake of 142 +/- 11 and 60 +/- 4 mumol X g-1 X h-1 were calculated for periportal and pericentral regions, respectively. Infusion of 2 mM lactate increased oxygen uptake by 71 mumol X g-1 X h-1 in periportal regions and by 29 mumol X g-1 X h-1 in pericentral areas of the liver lobule. Since the stoichiometry between glucose production and extra oxygen uptake is well-established, rates of glucose production in periportal and pericentral regions of the liver lobule were calculated from local changes in rates of oxygen uptake for the first time. Maximal rates of glucose production from lactate (2 mM) were 60 +/- 7 and 25 +/- 4 mumol X g-1 X h-1 in periportal and pericentral zones of the liver lobule, respectively. The lactate concentrations required for half-maximal glucose synthesis were similar (0.4-0.5 mM) in both regions of the liver lobule in the presence or absence of epinephrine (0.1 microM). In the presence of epinephrine, maximal rates of glucose production from lactate were 79 +/- 5 and 59 +/- 3 mumol X g-1 X h-1 in periportal and pericentral regions, respectively. Thus, gluconeogenesis from lactate predominates in periportal areas of the liver lobule during perfusion in the anterograde direction; however, the stimulation by added epinephrine was greatest in pericentral areas. Differences in local rates of glucose synthesis may be due to ATP availability, as a good correlation between basal rates of O2 uptake and rates of gluconeogenesis were observed in both regions of the liver lobule in the presence and absence of epinephrine. In marked contrast, when livers were perfused in the retrograde direction, glucose production was 28 +/- 5 mumol X g-1 X h-1 in periportal areas and 74 +/- 6 mumol X g-1 X h-1 in pericentral regions.(ABSTRACT TRUNCATED AT 400 WORDS)     

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) reduces lipoprotein lipase activity in the adipose tissue of the guinea pig

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) administered to young male guinea pigs at a dose of 1 microgram/kg (single intraperitoneal injection) caused a large reduction in adipose tissue lipoprotein lipase (LPL) activity. This effect occurred rapidly; a 70% decrease was noticed after 24 hour and 80% of LPL activity was lost by 48 hours when the serum triglyceride levels increased to 175% of control levels. LPL is known to play an important role in controlling the amount of free fatty acids supplied to adipose tissues. Administration of a large dose of glucose to fasted guinea pigs, which have shown a similar weight loss, but less LPL loss than TCDD-treated animals, had the effect of elevating their adipose LPL levels back to a near normal level, whereas the same treatment caused no significant increase in the LPL levels of TCDD-treated animals. Evidence indicates that the TCDD-caused decline in LPL activity is irreversible. As a consequence, the affected guinea pigs are incapable of responding to changes in nutritional status.     

In vivo and in vitro studies on the appearance of LHRH neurons in the hypothalamus of perinatal rats

Summary Ontogenetic development of LHRH-containing neurons was studied by fluorescence and enzyme immunohistochemistry in rats. In in vitro studies, the tissues of the septal-chiasmatic and mediobasal hypothalamic areas of fetal rats on day 16.5 or 18.5 of gestation were trypsinized separately for dissociation of the neural cells, and cultured for several days. Immunopositive reaction against LHRH was first detected in nerve cells derived from both areas of the hypothalamus of the fetuses on days 16.5 and 18.5 of gestation, after 8 and 6 days culture, respectively. The cells were small, and seemed to be bipolar in morphology indicating an axon and arborized dendrites. Immunopositive material occurred in the cell soma as well as in the cellular processes. In in vivo studies, immunopositive material, possibly deposited in nerve fibers, appeared first in OVLT and simultaneously in the external layer of the median eminence of fetuses on day 20.5 of gestation. The immunoreactive fibers increased in number in both parts with development, especially after birth in the median eminence. No immunopositive material was detected within any neural cell bodies nor in the cytoplasm of any ependymal cells.This work was financed by the Ministry of Education, Japan. No. 257008. We would like to thank Dr. Katsuhiko Saito (Department of Surgery, Tokushima University) for his kind advice on the preparation of the antibody used for the immunofluorescence study.     

Resonance Raman spectra of riboflavin and its derivatives in the bound state with egg riboflavin binding proteins

The resonance Raman spectra of riboflavin (RF) and its derivatives, including 3-deuterated (3-D RF), 3-methyl (3-CH3 RF), 3-carboxymethyl (3-CH2COOH RF), and 7,8-dichlororiboflavins (7,8-Cl RF), in H2O and D2O were observed in the 700-1700 cm-1 region. The fluorescence problem of riboflavin was overcome by complex formation of riboflavin with riboflavin binding proteins. The observed frequencies of Raman lines of RF are in good agreement with those of glucose oxidase obtained by Spiro et al. by the resonance CARS method, although the present spectral range is extended to much lower frequency with a higher signal-to-noise ratio than that for glucose oxidase. The observed Raman lines were assigned to the individual ring modes of isoalloxazine on the basis of the Raman spectra of appropriate model compounds such as uracil, pyrazine, and o-xylene. The 1253 cm-1 line of RF was shifted to ca. 1300 cm-1 for 3-D RF, 3-CH3 RF, and 3-CH2COOH RF, and accordingly can be assigned to the CN stretching mode of Ring III. The 1632 cm-1 line of RF was shifted for 7,8-Cl RF and was assigned to a Ring I mode. No Raman line mainly due to C = O stretching mode was observed in the present resonance Raman spectra.