Dry Ice-acetone Research Articles

A simple method for the preparation of 2,4,6-trichloroborazine and evaluation of 1H-NMR spectra

ABSTRACT: The compound 2,4,6-trichloroborazine (TCB) is known as a valuable precursor to prepare boron nitride and various derivatives of borazine. Difficulties in the preparation of TCB via conventional methods on the one hand, and its importance for the material science, on the other hand, were our motivation to modify the Brown’s method for TCB synthesis. So far, all reports on the synthesis procedure of TCB are based on the use of gaseous BCl3 with NH4Cl powder at 110-130 °C in chlorobenzene requiring complicated low-temperature equipment such as a cold finger condenser, and dry ice-acetone reflux condenser to retain BCl3 gas in the reaction flask. According to our study, using BCl3 solution in n-hexane was more convenient than using gaseous BCl3. The reaction of BCl3 solution with NH4Cl was carried out without loss of BCl3. Furthermore, the reduction in the reflux temperature from 130 °C in some previous studies to 86 °C in our study, was a valuable advantage, leading to the release of the coordinated BCl3 from the adduct complex [CH3CN·BCl3] gradually and the prevention of thermal decomposition of TCB, unwanted polymerization reactions and the formation of undesirable products, resulting in the increased reaction yield. TCB was fully characterized by ATR-FTIR, 11B-, 14N- and 1H-NMR spectroscopic methods. 1H-NMR spectra of TCB were performed for the first time at various temperatures to elucidate the quadrupolar effect of nitrogen, which led to evaluate the splitting of the proton resonance of the 14N-H bond by coupling with the 14N quadrupole nucleus. The broad peak observed at room temperature was clearly split into a triplet at 100 °C due to the long TqN and fast molecular motion of TCB molecules. The 14N-NMR spectrum was also shown for the first time as a broad signal at δ = –271.3 ppm (h1/2 = 186 Hz).

Freeze-thaw system for thermostable β-Galactosidase isolation from Gedong Songo Geobacillus sp. isolate

The effective isolation of intracellular enzymes from thermophilic bacteria is challenging because of their sturdy membrane. On the other hand, the low-cost and nontoxic method is essential for industrial food enzymes. The freeze-thaw cycles using acetone-dry ice as a frozen system was studied for efficient isolation of thermostable b-galactosidase from Geobacillus sp. dYTae-14. This enzyme has been known for application in the dairy industry to reduce the lactose content. In this study, the freeze-thaw method was performed with cycle variations 3, 5, and 7 cycles. Acetone-dry ice (-78°C) is used as a frozen system and boiling water for thawing. The b-galactosidase activity was assayed using ortho-Nitrophenyl-β-galactoside (ONPG) as substrate and protein content determined with the Lowry method. The results show that the most effective freeze-thaw is five cycles. The enzyme’s highest specific activity is 3610.13 units/mg proteins at 40-60 % ammonium sulfate saturation, with a purity value of 2.52.

Effect of production factors on muscle fiber type and dimensions in the m. semimembranosus of crossbred steer carcasses

The muscle fibers that have been examined in the study were affected by three different controlled factors: steroids, ractopamine and residual feed intake (RFI). By examining the effects of the controlled factors on cattle’s muscle fibers, it can be determined if they affect different meat properties, such as meat toughness, collagen solubility and muscle fiber quality. The research had been done specifically with m. semimembranosus (SM) of crossbred steers. Although some may be concerned with the health effects of steroids and other materials, no negative effects to the health of the cattle were observed after the use of steroids. This is because the hormones being introduced into the cattle’s body already exist in the animal. In addition, the same concept applies to humans who consume the meat, preventing harm the people who consume it. For this study, 48 crossbred angus steers were used, 12 for each of the different treatment groups. The control group consisted of no steroids and no ractopamine. The second group was not treated with steroid but with ractopamine. The third group was treated with steroids but no ractopamine. Finally, the fourth group was treated with both, the steroids and the ractopamine. For each SM muscle, 1-inch thick steaks were cut and from those steaks, 1cm3 cubes were cut. These cubes were frozen in dry ice acetone until they are ready to be sectioned. Cubes are placed in the cryostat and sliced into serial sections of 10µm. These serial sections are then mounted onto dry slide glass and stored in a freezer at -80ºC until they are to be stained. The staining process helps to identify the different types of muscle fibers in the samples. From the muscle fiber types, the average sizes of each muscle fiber is calculated to identify inconsistencies among the different treatment groups. Conclusions will be drawn based on the inconsistencies found (if any).

Effect of pretreatment with a tyrosine kinase inhibitor (PP1) on brain oedema and neurological function in an automated cortical cryoinjury model in mice

Cerebral oedema is a significant cause of morbidity in neurosurgical practice. To our knowledge, there is no ideal drug for prevention or treatment of brain oedema. Based on the current understanding of the pathogenesis of brain oedema, tyrosine kinase inhibitors could have a role in reducing brain oedema but preclinical studies are needed to assess their effectiveness. We evaluated the role of pretreatment with 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP1), an Src tyrosine kinase inhibitor, in reducing cerebral oedema and preserving neurological function measured 24hours after an automated cortical cryoinjury in mice. Sixteen adult male Swiss albino mice were subjected to an automated cortical cryoinjury using a dry ice–acetone mixture. The experimental group (n=8) received an intraperitoneal injection of PP1 dissolved in dimethyl sulfoxide (DMSO) at a dose of 1.5mg/kg body weight 45minutes prior to the injury. The control group (n=8) received an intraperitoneal injection of DMSO alone. A further eight mice underwent sham injury. The animals were evaluated using the neurological severity score (NSS) at 24hours post-injury, after which the animals were sacrificed and their brains removed, weighed, dehydrated for 48hours and weighed again. The percentage of brain water content was calculated as: {[(wet weight – dry weight)/wet weight] × 100}. The mean (standard deviation, SD) NSS was 11.7 (1.8) in the experimental group and 10.5 (1.3) in the control group (p=0.15). The mean (SD) percentage water content of the brain was 78.6% (1.3%) in the experimental group and 77.2% (1.1%) in the control group (p=0.03). The percentage water content in the experimental and control groups were both significantly higher than in the sham injury group. The immediate pre-injury administration of PP1 neither reduced cerebral oedema (water content %) nor preserved neurological function (NSS) when compared to a control group in this model of cortical cryoinjury.

Packed-Bed Catalytic Cracking of Oak-Derived Pyrolytic Vapors

Catalytic upgrading of pyrolysis vapors derived from oak was carried out using a fixed-bed catalytic column at 425 °C. The vapors were drawn by splitting a fraction from the full stream of vapors produced at 500 °C in a 5 kg/h bench-scale fast pyrolysis reactor system downstream from the cyclone separator. The placement of the fixed-bed column was varied within the condenser train to determine the effect of temperature, residual water, solids, and oxygenated components of the approach vapor stream on the upgraded product quality. The upgraded liquid was collected by immediate contact with a dry ice acetone bath, complimented by a secondary collection system comprised of a methanol spray condenser. Quantitative gas chromatography/mass spectroscopy (GC/MS) analysis of the recovered liquid showed a substantial decrease of oxygen-containing species with a significant increase in carbon-rich (≥C6) aromatic hydrocarbons. The extent of deoxygenation was location-specific and dependent upon temperature and the re...

A rubber transfer gasket to improve the throughput of liquid–liquid extraction in 96-well plates: Application to vitamin D testing

Background The unmitigated rise in demand for the assessment of vitamin D status has taxed the ability of clinical mass spectrometry laboratories to preserve turn-around times. We aimed to improve the throughput of liquid–liquid extraction of plasma/serum for the assay of 25-hydroxy vitamin D. Methods We designed and fabricated a flexible rubber gasket that seals two 96-well plates together to quantitatively transfer the contents of one plate to another. Using the transfer gasket and a dry-ice acetone bath to freeze the aqueous infranatant, we developed a novel liquid–liquid extraction workflow in a 96-well plate format. We applied the technology to the mass spectrometric quantification of 25-hydroxy vitamin D. Results Cross-contamination between wells was ≤0.13%. The interassay imprecision over 132 days of clinical implementation was less than 10%. The method compared favorably to a standard liquid–liquid extraction in glass tubes (Deming slope = 1.018, S x|y = 0.022). The accuracy of the assay was 102–105% as assessed with the recently released control materials from NIST. Conclusions The development of a plate-sealing gasket permits the liquid–liquid extraction of clinical specimens in a moderate-throughput workflow and the reliable assay of vitamin D status. In the future, the gasket may also prove useful in other sample preparation techniques for HPLC or mass spectrometry.

Generation of volatile cobalt species by UV photoreduction and their tentative identification

Generation of volatile cobalt carbonyl was accomplished by exposing a solution of inorganic cobalt, containing low molecular weight organic acids, to UV irradiation. The gaseous products were removed from the liquid phase in a gas–liquid separator and transported to a Pyrex U-tube cryogenic trap. Following cryocondensation of the products using a dry ice–acetone bath, the U-tube was placed in a hot water bath (35–40 °C) and the trapped species were introduced to an ICP-MS for estimation of the efficiency of generation. Alternatively, the gas phase containing the Co species was sampled with a gastight syringe and injected into a GC-MS for species identification. The product, which is stable for periods in excess of 60 minutes, is tentatively identified as a carbonylated cobalt species, generated with an efficiency of 0.3–0.4%

Femtomolar Transition State Analogue Inhibitors of 5′-Methylthioadenosine/S-Adenosylhomocysteine Nucleosidase from Escherichia coli

Escherichia coli 5'-methylthioadenosine/S-adenosyl-homocysteine nucleosidase (MTAN) hydrolyzes its substrates to form adenine and 5-methylthioribose (MTR) or S-ribosylhomocysteine (SRH). 5'-Methylthioadenosine (MTA) is a by-product of polyamine synthesis and SRH is a precursor to the biosynthesis of one or more quorum sensing autoinducer molecules. MTAN is therefore involved in quorum sensing, recycling MTA from the polyamine pathway via adenine phosphoribosyltransferase and recycling MTR to methionine. Hydrolysis of MTA by E. coli MTAN involves a highly dissociative transition state with ribooxacarbenium ion character. Iminoribitol mimics of MTA at the transition state of MTAN were synthesized and tested as inhibitors. 5'-Methylthio-Immucillin-A (MT-ImmA) is a slow-onset tight-binding inhibitor giving a dissociation constant (K(i)(*)) of 77 pm. Substitution of the methylthio group with a p-Cl-phenylthio group gives a more powerful inhibitor with a dissociation constant of 2 pm. DADMe-Immucillins are better inhibitors of E. coli MTAN, since they are more closely related to the highly dissociative nature of the transition state. MT-DADMe-Immucillin-A binds with a K(i)(*) value of 2 pm. Replacing the 5'-methyl group with other hydrophobic groups gave 17 transition state analogue inhibitors with dissociation constants from 10(-12) to 10(-14) m. The most powerful inhibitor was 5'-p-Cl-phenylthio-DADMe-Immucillin-A (pClPhT-DADMe-ImmA) with a K(i)(*) value of 47 fm (47 x 10(-15) m). These are among the most powerful non-covalent inhibitors reported for any enzyme, binding 9-91 million times tighter than the MTA and SAH substrates, respectively. The inhibitory potential of these transition state analogue inhibitors supports a transition state structure closely resembling a fully dissociated ribooxacarbenium ion. Powerful inhibitors of MTAN are candidates to disrupt key bacterial pathways including methylation, polyamine synthesis, methionine salvage, and quorum sensing. The accompanying article reports crystal structures of MTAN with these analogues.

EFFECT OF FROZEN TEMPERATURE ON AUTOXIDATION AND AGGREGATION OF BLUEFIN TUNA MYOGLOBIN IN SOLUTION

The effect of frozen storage temperatures in the range of -3C to -80C on the autoxidation and aggregation of bluefin tuna oxy- and deoxy-myoglobin (Mb) in solution was examined. Mb solution was kept frozen at various temperatures after prefreezing by dry ice-acetone and examined for autoxidation rate constant and aggregation ratio. The temperature-dependency of these two parameters showed the highest values at -10C with both oxy or deoxy forms. The autoxidation rate constant of deoxyMb was higher than that of oxyMb between -6C and -20C, while the reverse trend was observed at lower temperatures. These results suggest that the structure of Mb was perturbed through freezing resulting in promotion of the autoxidation and aggregation of Mb simultaneously. These results could explain the observation that deoxyMb in the inner portion of tuna flesh discolors faster than oxyMb in the surface portion in the temperature range of -3C and -20C.

Histological and Mucin Histochemical Study of Colon Ulcers Induced in Rats by a Rapid Freezing Method

The purpose of this study was to make a new experimental colon ulcer model that is independent on chemicals and to assess the ulcerations and subsequent changes at various time points. Histological and mucin histochemical assessment of colon ulcers and associated regeneration induced in Wistar rats using a rapid freezing method was performed. Male rats were fasted 48 hours prior to surgery. A lower midline incision of the abdominal wall was made and a stainless steel rod, kept in dry ice-acetone, was applied to the serosal surface of the colon for 4 seconds, twice, with a 5 seconds interval. The rats were sacrificed at various time points thereafter. Histological examination showed the ulcers to be accompanied by acute inflammation, and healing was complete in almost all cases at day 24. In the ascending colon, sulfomucin was found in goblet cells in the upper half of the normal crypts, and sialomucin was found in the lower half. In the descending colon, sulfomucin was apparent almost throughout normal crypts remote from ulcers. Sialomucin dominant regenerative crypts were found near areas of ulceration in both the ascending and descending colon. Thus, the simple freezing method can produce colon ulcers, and the sialomucin that was predominant in regenerative crypts may have an important role for regeneration.

Novel catalysts for advanced hydroprocessing: transition metal phosphides

The field of catalysis is continuously invigorated by the discovery of new catalytic materials. Transition metal phosphides represent one such group of compounds that have recently been shown to have excellent activity for hydrodesulfurization (HDS) and hydrodenitrogenation (HDN). This is an important area, as continuous decline in the quality of petroleum feedstocks and increasingly stringent environmental regulations have made the removal of sulfur a paramount problem in the refining industry. The structure of the metal-rich phosphides is based on trigonal prisms, which can well accommodate the relatively large phosphorus atoms. The prisms are similar to those in sulfides, but phosphides do not take on layered structures and are metal conductors, not insulators or semiconductors. Lack of layers leads to a more isotropic crystal morphology and potentially better exposure of surface metal atoms to fluid phase reactants. Phosphides can be prepared readily in bulk and supported forms by reduction of phosphate precursors. The catalytic activity of the phosphides for dibenzothiophene HDS and quinoline HDN at 643 K and 3.1 MPa followed the order Fe2P<CoP<MoP<WP<Ni2P. The best catalyst, Ni2P/SiO2, had better activity in hydroprocessing than a commercial Ni–Mo–S/Al2O3 (HDS 98% vs 78% and HDN 80% vs 43%), based on equal sites loaded in the reactor. The sites were titrated by CO chemisorption at room temperature for the phosphides and by pulse O2 chemisorption at dry-ice acetone temperatures for the sulfide. The Ni2P/SiO2 also compared favorably with a commercial Co–Mo–S/Al2O3 catalyst (Ketjenfine 756) using a real feed at 593 K and 3.9 MPa. On the basis of equal weights loaded in the reactor, the Ni2P was again found to have higher performance than the sulfide (HDS 85% vs 80%). Studies of Ni2P/SiO2 catalysts with varying Ni/P ratios indicated that the principal phase remained Ni2P, but that the crystallite size decreased with increasing P content as a result of facilitated contact on the surface between the Ni component and the phosphorus reagent. Activity measurements on these samples indicated that HDS was structure-insensitive, but that HDN was structure-sensitive. Extended X-ray absorption fine structure measurements indicated that the surface formed a phospho sulfide after reaction. The mechanism of HDN was investigated using a series of molecular probes of varying structure. The reactivity order for pentylamines indicated that CNH2 bond scission proceeded by a β-hydride elimination mechanism, similar to that occurring on sulfides. The reactivity order of substituted piperidines indicated that ring-opening proceeded by CN bond scission and involved an α-carbon, and thus was different from the pathway on sulfides, which again appeared to involve a β-hydride elimination. This difference may account for the higher activity of phosphides over sulfides.

Determination of ionized magnesium in erythrocytes using a potentiometric analyzer

A simple method for the determination of ionized magnesium in erythrocytes using a potentiometric analyzer Microlyte 6 (Kone, Finland) was investigated. A set of aqueous solutions containing electrolytes at the same concentration as in erythrocytes in Tris/TES buffer (pH 7.2) was used as standards. The erythrocyte cell membranes were destroyed using three different methods: (A) HCl solution of pH 3.5, (B) dry ice–acetone mixture, (C) ultrasound. The result did not differ significantly between methods B and C (Student’s test, α=0.01). The results were compared with those obtained using the 31 P nuclear magnetic resonance method ( 31 P NMR). The dissociation constant of MgATP was calculated from the 31 P NMR results. The results obtained from potentiometry and 31 P NMR did not differ significantly (Student’s test, α=0.01). Total magnesium concentration was determined using atomic absorption spectrometry (AAS).

Syneretic Response to Pressure in Bovine and Rhesus Monkey Vitreous

Excised bovine and rhesus monkey vitreous samples were subjected to different hydrostatic pressures (0.03, 1 and 2atm) for 12hr. At the end of the experiment the samples were frozen in a −70°C dry ice-acetone bath and thereafter maintained in frozen conditions. Differential scanning calorimetry (DSC) was used to obtain freezable water (FW) content. The total water content of the samples was obtained by thermogravimetric (TGA) techniques. The non-freezable water (NFW) content was obtained as a difference between total and freezable water contents. The total water content did not show a trend in pressure dependence, neither in bovine nor in rhesus monkey vitreous samples. However, the free water/bound water ratio, i.e. FW/NFW, increased with increasing pressure in vitreous samples of both species. The trend of the average FW/NFW values as a function of hydrostatic pressure implies a simple syneretic response, i.e. the conversion of bound (non-freezable) water to free water as pressure was increased.

Matrix effect in the cryotrapping of gas samples in the ppbv range

A gas mixture containing ppbv concentrations of volatile chlorinated and aromatic hydrocarbons was prepared in helium, nitrogen, or argon in a plastic (Nalophan) bag, in which it was found the mixture could be safely stored for several days. Samples of the mixture were subsequently analyzed by gas chromatography after direct cryotrapping in an empty metal U-tube. The cooling medium used was liquid nitrogen, liquid argon, or a dry ice-acetone slurry (197 K). The efficiency of cryotrapping in liquid nitrogen was over 90% when the aromatic hydrocarbon mixture was prepared in the helium matrix, but between 50 and 70% when it was prepared in nitrogen or argon, the recovery from the argon matrix being somewhat higher. The poor recovery in nitrogen at 77 K and argon at 87 K was explained by the reduced rate of diffusion to the wall owing to mist or aerosol formation. At 197 K condensation was negligible if the partial pressures were lower than the saturated vapor pressures.

Role of nitric oxide in regulating the ductus arteriosus caliber in fetal rats.

The role of nitric oxide (NO) on the ductus arteriosus (DA) patency was examined in fetal rats at various stages of gestation. N(G)-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg, i.p.), an NO synthase (NOS) inhibitor, or indomethacin (3 mg/kg, p.o.), a cyclooxygenase inhibitor, was administered at 3 hr before cesarean section to pregnant rats ranging from day 17 to day 21 of gestation. Dams were decapitated and the fetuses were obtained by cesarean section. The fetuses were rapidly frozen in an acetone-dry ice mixture. Using rapid-freezing and shaving methods, the calibers of the DA and pulmonary artery were measured. The constrictive effect of L-NAME on the fetal DA caliber was stronger than that of indomethacin in 19-day-old and immature fetuses. In near-term fetuses, the constrictive effects of L-NAME were reduced, while indomethacin caused marked DA constriction. We conclude that endogenous NO may play a major role in regulating the patency of the DA in earlier fetal stages, while dilator prostaglandins may play a greater role in regulating the ductal patency in the near-term fetus.

Effects of TAK-044, a nonselective endothelin receptor antagonist, on the spontaneous and indomethacin- or methylene blue-induced constriction of the ductus arteriosus in rats.

We studied the effects of TAK-044, a nonselective endothelin (ET) receptor antagonist, on the indomethacin- or methylene blue-induced constriction of the ductus arteriosus (DA) in rats and compared them with the effects on spontaneous DA constriction. Injection of TAK-044 into 21-day-old fetuses in utero was performed through the uterine wall of laparotomized mother rats under light ether anesthesia. The fetuses were autopsied 3 hr after treatment with TAK-044 (10 mg/kg) in utero and simultaneous administration to the laparotomized mother rats of indomethacin (3 mg/kg, p.o.) or methylene blue (100 mg/kg, i.p.). In the second experiment, pregnant rats were decapitated on day 21 of gestation to obtain newborn rats by cesarean delivery. Newborn rats which were given TAK-044 (2, 10 mg/kg) immediately after or 1 hr before cesarean delivery were autopsied at various times after birth. In both experiments, pups were rapidly frozen in an acetone-dry ice mixture at autopsy to evaluate the DA constriction by the whole-body freezing and shaving method. TAK-044 injection into the fetus 3 hr before autopsy completely inhibited the DA constriction induced by maternal treatment with indomethacin or methylene blue. TAK-044 caused dose-dependent inhibition of the spontaneous closure of the DA after birth. The inhibitory effect was more pronounced in pups which were given TAK-044 in utero 1 hr before birth; however, the inhibitory effect was incomplete in newborn pups. These results, together with the previous finding that BQ-123, an ETA-specific receptor antagonist, inhibits the ductal constriction induced by oxygen in vitro [Coceani et al., 1992], indicate that the ETA receptor plays a significant role in the indomethacin- or methylene blue-induced DA constriction as well as in the spontaneous DA constriction after birth, and also indicate that the inhibition of ETA receptor by TAK-044 was more easily achieved in fetuses than in neonates.

Role of the nitric oxide-cGMP system in the regulation of ductus arteriosus patency in fetal rats.

The purpose of this study was to examine the role of the nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) system in the regulation of the ductus arteriosus (DA) patency in fetal rats. Pregnant rats were administered N(G)-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg, ip), an NO synthase (NOS) inhibitor; methylene blue (30, 50 and 100 mg/kg, ip), a soluble guanylate cyclase inhibitor; or indomethacin (3 mg/kg, po), a cyclooxygenase inhibitor, at various times before cesarean section. Dams were decapitated to obtain the fetuses by cesarean section, and fetuses were rapidly frozen in an acetone-dry ice mixture. Using rapid freezing and shaving methods, the calibers of the DA, pulmonary artery (PA) and descending aorta (Ao) were measured to evaluate the effects of treatment. L-NAME reduced the DA calibers to 86% of the initial values, but recovery to the control levels occurred 6 hr after the injection. Indomethacin decreased the DA calibers to 34% of the control values and sustained the DA constriction until 24 hr after the treatment. Methylene blue caused DA constriction to almost the same degree as indomethacin, but the levels normalized within 24 hr after the treatment. We conclude that L-NAME caused a slight constriction of the DA, whereas methylene blue and indomethacin caused marked constriction of the vessels, suggesting that the NO-cGMP system as well as prostaglandins contribute to the DA patency.

Sucrase-isomaltase and other brush border glycosidases in colorectal tumors

Sucrase-isomaltase (SI), trehalase (T) and lactase-beta-glucosidase (LG) activities were assessed histochemically in samples of colorectal adenomas (11 tubular, 12 tubulovillous, 10 villous) and 30 adenocarcinomas obtained by biopsy during colonoscopy or from specimens removed by surgical intervention. Small samples of tumor tissue, tissue of the transitional zone and of macroscopically normal mucosa were quenched in heptan cooled in an acetone-dry ice mixture. Cryostat sections, transferred to non-precooled slides and in some cases to semipermeable membranes, were dried and subjected to the histochemical reactions for SI, T and LG. Sucrose, 2-naphthyl, 6-Br-2-naphthyl, and 5-Br-4-Cl-3-indoxyl alpha-D-glucosides, trehalose, and 5-Br-4-Cl-3-indoxyl-beta-D-fucoside were used as substrates. Sections of jejunal biopsies with normal activities of brush border glycosidases were used as controls. From samples of 5 adenomas, 5 adenocarcinomas and collected rests of jejunal biopsies with a normal finding 10% (w/vol) homogenates in 2% Triton X-100 were prepared. Homogenates were frozen and thawed 3 times and their supernatants subjected to isoelectric focusing on polyacrylamide gel plates. Zymograms were developed with the same methods as for the detection of alpha-glucosidases in sections. In no colorectal tumor LG was present. SI was found in 70% adenocarcinomas, 50% villous, 25% tubulovillous and 19% tubular adenomas when the method with sucrose, glucose oxidase-peroxidase and 3,3'-diaminobenzidine was used. Hardly discernible traces of activity were found in tumors with azo-coupling reactions applied at pH 5, 6 and 6.5. No reaction was detected with the indigogenic method applied at pH above 6.0. However, jejunal biopsies displayed very strong reactions confined to the brush border of enterocytes under the same conditions. A strongly positive reaction was seen in 7 of 12 tumors investigated recently when the indigogenic reaction was applied at pH below 6.0 (particularly at pH 5.0). In this case the deposition of indigo was due to membrane and lysosomal alpha-glucosidases of the tumor cells and lysosomal alpha-glucosidase of macrophages and leukocytes. These findings were corroborated by zymograms. T was detected in the same tumors as SI; its activity was lower, however. SI activity in colorectal tumors is a useful, but not general marker of these tumors.

Myosin light chain phosphorylation in contraction of gastric antral smooth muscle from neonate and adult rabbits.

The decreased contractility of gastric antral smooth muscle in the neonate has been attributed to reduced levels of activator calcium. It is generally accepted that calcium-dependent myosin light chain phosphorylation (MLCP) is the key step in the initiation of force development in smooth muscle. In this study, we investigated the relationship between MLCP and force development in gastric antral smooth muscle from neonatal (4-6 d old) and adult rabbits. We tested the hypothesis that the reduced force development of circular smooth muscle from the neonate would be accompanied by decreased levels of MLCP, as compared with data from adult animals. Full thickness muscle strips oriented parallel to the circular muscle layer were examined for their contractile response to acetylcholine (ACh) (10(-8) M to 10(-3) M) or 10(-4) M ACh only. In the latter study, tissues were rapidly frozen in a dry ice-acetone slurry for subsequent MLCP determination. MLCP was determined at times corresponding to 5, 10, 15, 30, and 60 s of stimulation. For each age group, maximal active force developed at an ACh concentration of 10(-4) M and was significantly greater in tissues from adults (1.86 +/- 0.24 N/m2, adult; 0.95 +/- 0.05 N/m2, neonate; p < 0.05). In contrast, no significant differences were observed with respect to basal or agonist-stimulated levels of MLCP. The data suggest that factors other than levels of MLCP contribute to the reduced force-generating capacity of antral smooth muscle from the neonate.

Role of myosin phosphorylation and [Ca2+]i in myogenic reactivity and arteriolar tone.

The aim of this study was to define the relationship between intraluminal pressure, intracellular calcium concentration ([Ca2+]i), and myosin light-chain (MLC) phosphorylation in isolated arterioles exhibiting myogenic tone. Cremaster muscles were removed from anesthetized rats, and arterioles (approximately 100-microns diam) were dissected from surrounding tissues and cannulated on glass pipettes. Vessels were warmed to 34 degrees C and initially pressurized to 70 mmHg in the absence of intraluminal flow. For [Ca2+]i measurements, vessels were loaded with 5 microM fura 2, and fluorescence emitted by excitation at 340 and 380 nm was measured. Data were considered in terms of changes in the fluorescence ratio (340/380 nm) and collected at steady-state intraluminal pressures between 30 and 170 mmHg. For measurement of MLC phosphorylation, vessels were frozen in acetone-dry ice followed by sonication in homogenizing buffer. Homogenates were separated by two-dimensional gel electrophoresis, and proteins were visualized by silver staining. MLC phosphorylation was quantitated photodensitometrically, and results are expressed as percent total 20-kDa MLC. Increasing intraluminal pressure resultedin significant constriction with increased [Ca2+]i and MLC phosphorylation. For example, the fluorescence ratio was 0.80 +/- 0.04 at 30 mmHg compared with 1.02 +/- 0.05 at 120 mmHg (n = 7 vessels); corresponding MLC-phosphorylation values were 27.7 +/- 1.6 and 39.6 +/- 3.0% (n = 6). MLC phosphorylation in arterioles superfused with 0 mM Ca(2+)-2 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was 8.5 +/- 0.7%.(ABSTRACT TRUNCATED AT 250 WORDS)