Carbonic anhydrase immobilized on hollow fiber membranes using glutaraldehyde activated chitosan for artificial lung applications.

Extracorporeal CO2 removal from circulating blood is a promising therapeutic modality for the treatment of acute respiratory failure. The enzyme carbonic anhydrase accelerates CO2 removal within gas exchange devices by locally catalyzing HCO3 (-) into gaseous CO2 within the blood. In this work, we covalently immobilized carbonic anhydrase on the surface of polypropylene hollow fiber membranes using glutaraldehyde activated chitosan tethering to amplify the density of reactive amine functional groups for enzyme immobilization. XPS and a colorimetric amine assay confirmed higher amine densities on the chitosan coated fiber compared to control fiber. Chitosan/CA coated fibers exhibited accelerated CO2 removal in scaled-down gas exchange devices in buffer and blood (115% enhancement vs. control, 37% enhancement vs. control, respectively). Carbonic anhydrase immobilized directly on hollow fiber membranes without chitosan tethering resulted in no enhancement in CO2 removal. Additionally, fibers coated with chitosan/carbonic anhydrase demonstrated reduced platelet adhesion when exposed to blood compared to control and heparin coated fibers.

Bromocriptine reverses the inhibitory effect of macrophages on human sperm motility.

OBJECTIVE: To determine the effects of PRL suppression on the activation of murine peritoneal macrophages and their subsequent effects on human sperm motility. DESIGN: Laboratory study. INTERVENTIONS: Mice were treated with subcutaneous implants of 2.5 mg bromocriptine pellets 7 days before bacillus of calmette and guerin (BCG), a strain of Mycobacterium bovis, injection for activation of macrophages. Bromocriptine treatment, which significantly suppressed circulating PRL levels, continued through the day of peritoneal macrophage collection. Macrophages were subsequently cultured for 4 days and culture supernatant was collected. MAIN OUTCOME MEASURES: Human sperm were incubated for 4 hours in the presence of culture medium or culture supernatant from control and treated mice. Motion analysis was performed at 0, 2, and 4 hours. RESULTS: A significant decrease in human sperm motility was observed in the presence of culture supernatant from activated murine peritoneal macrophages. After 4 hours of incubation, sperm motility decreased from 69% +/- 3% in the nonactivated macrophage control group to 37% +/- 6% in the BCG- activated macrophage group. The suppressive effect of soluble products of activated macrophages on human sperm motility was reversed when mice were rendered hypoprolactinemic with bromocriptine. Motility after 4 hours was 56% +/- 3% in the BCG-bromocriptine group. Simultaneous administration of bromocriptine and PRL (100 ng per mouse daily) restored the inhibitory effect of soluble products of activated macrophages on sperm motility (36% +/- 5% motile). CONCLUSIONS: PRL may modulate the deleterious effects of activated macrophages on human sperm motility, thereby suggesting novel and useful methods for the modification of the immune response in early reproduction.

Immunohistochemically demonstrated androgen receptor expression in the rat prostate during carcinogenesis induced by 3,2'-dimethyl-4-aminobiphenyl with or without testosterone.

A polyclonal antibody against rat androgen receptor was used to investigate its expression during 3,2'-dimethyl-4-aminobiphenyl (DMAB)-induced prostate carcinogenesis in the rat. Fixation in cold-acetone provided better observation of specific nuclear staining than 10% buffered formalin. Normal epithelial cells and atypical hyperplasias in all lobes were positive. Ventral prostate carcinomas were also strongly positive for androgen receptor (AR) but most invasive carcinomas arising from the dorsolateral prostate and seminal vesicles, which were induced by DMAB plus testosterone propionate, were AR negative. Atypical hyperplasias of those organs were all positive. These findings suggest that ventral carcinomas are androgen dependent but carcinomas in other lobes become independent during progression.

Single and repeated environmental stress: effect on plasma oxytocin, corticosterone, catecholamines, and behavior.

Rat studies were done to further characterize an environmental model of stress designated shaker stress (SS). Plasma oxytocin (OT), corticosterone (CS), norepinephrine (NE), and epinephrine (E) were measured before and after 5 or 30 min of SS applied one time or applied 10 times over a 2-week period. The major findings were partial adaptation of plasma E within 30 min of acute SS, adaptation of plasma CS baselines but not responses to chronic SS, and complete adaptation of plasma OT responses to chronic SS. Poststress behavior during chronic SS was affected in the following ways: freezing time habituated, defecation and rearings increased, and grooming and teeth chattering remained relatively constant. The results show that SS produces consistent patterns of hormonal and behavioral responses; some aspects of the patterns are similar to those elicited by other environmental stresses, whereas some aspects are unique to SS. We conclude that rats do not adapt to repeated SS but rather that most hormonal and behavioral defense mechanisms are renewable on a daily basis.

Influence of L-methionine-deprived total parenteral nutrition with 5-fluorouracil on gastric cancer and host metabolism.

AIM: To investigate the influence of L-methionine-deprived total parenteral nutrition with 5-FU on gastric cancer and host metabolism. METHODS: N-methyl-N'-nitro-nitrosoguanidine (MNNG) induced gastric cancer rats were randomly divided into four groups: Met-containing TPN group (n=11), Met-deprived TPN group (n =12), Met-containing TPN+5-FU group (n=11) and Met-deprived TPN+5-FU group (n=12). Five rats in each group were sacrificed after 7 days of treatment and the samples were taken for examination. The remaining rats in each group were then fed separately with normal diet after the treatment until death, the life span was noted. RESULTS: The tumors were enlarged in Met-containing group and shrank in Met-deprived group markedly after the treatment. The DNA index (DI) of tumor cells and the body weight (BW) of rats had no significant change in the two groups, however, the ratio of tumor cells'S phase was increased. The ratio of G2M phase went up in Met-containing group, but down in Met-deprived group. In the other two groups that 5-FU was added, the BW of rats, and the diameter of tumors, the DI of tumor cells, the S and G2M phase ratio of tumor cells were all decreased, particularly in Met-deprived plus 5-FU group. Pathological examination revealed that the necrotic foci of the tumor tissue increased after Met-deprived TPN treatment, and the nucleoli of tumor cells enlarged. In MetTPN+5-FU group, severe nuclear damage was also found by karyopyknosis and karyorrhexis, meanwhile there was slight degeneration in some liver and kidney cells. The serum free Met and Cysteine decreased markedly (P<0.001), while other amino acids, such as serum free serine and glutamine increased significantly (P<0.005). All the rats died of multiple organ failure caused by cancer metastasis. The average survival time was 18.6 days in Met-containing TPN group, 31 days in Met-deprived TPN group, 27.5 days in Met-containing TPN+5-FU group, and 43 days in Met-deprived TPN+5-FU group (P<0.05). CONCLUSION: Met-deprived TPN causes methionine starvation of tumor cells, and can enhance the anti-tumor effect of 5-FU and prolong the life span of gastric cancer bearing rats.

Activation of cellular oncogenes in human diploid cell strain (KMB-13) cells by scooter exhaust particulate matter emissions.

Scooter exhaust particulate matter emissions were found to be mutagenic and to induce potential carcinogenicity. To further explore the mechanisms of mutagenicity and potential carcinogenicity of scooter exhaust particulate matter emissions, immunohistochemistry assays were carried out to detect the expression of some oncogenes and tumor suppressor genes in human diploid cell strain (KMB-13) cells, which were morphologically transformed by the organic extracts of scooter exhaust particulate matter emissions. An ABC diagnostic kit was used to investigate the expression of c-myc, p21, p53, and p16 proteins in the transformed cells. The c-myc and p21 proteins showed marked positive staining compared to control. The data suggest that the mutagenicity and potential carcinogenicity of the scooter exhaust particulate matter emissions correlate with the activation of some cellular oncogenes.

Study on the mutagenicity of diesel exhaust particles.

The mutagenicity of diesel exhaust particles (DEP) was studied by using Salmonella typhimurium strains TA98 and TA100 in vitro and mice micronucleus in vivo test. DEP from six kinds of medium and heavy-duty diesel vehicles, which were made in China and imported, were tested. The vehicles were operated under free accelerating condition. The results showed that the DEP contained mutagenic activity. An increase in the number of the Salmonella TA98 was observed in the presence and especially in the absence of S9 mix. Positive results were also obtained from mice micronucleus assay. The frequency of mice bone marrow micronucleated polychromatic erythrocytes (M PCE) was increased and it showed a definite dose-response relationship. Comparing the different types of the vehicles, we found that the mutagenicity of DEP from domestic made vehicles was stronger than that from the imported ones.

Comparison of the mutagenicity of exhaust emissions from motor vehicles using leaded and unleaded gasoline as fuel.

While unleaded gasoline has the advantage of eliminating lead from automobile exhaust, its potential to reduce the exhaust gas and particles, merits further examination. In the present studies, the concentrations of hydrocarbons (HC) and carbon mono-oxides (CO) in emissions were analyzed on Santana engine Dynamometer under a standard test cycle, and total exhaust particles were collected from engines using leaded and unleaded gasoline. It was found that unleaded gasoline reduced the emissions of CO and HC, and decreased the quantity of vehicle exhaust particulate matters by 60%. With the unleaded gasoline, only 23 kinds of organic substances, adsorbed in the particles, were identified by gas chromatography/mass spectrometer (GC/MS) while 32 components were detected using the leaded gasoline. The results of in vitro Salmonella/microsomal test and micronucleus induction assay in CHL cells indicated that both types of gasoline increased the number of histidine-independent colonies and the frequencies of micronucleus induction; no significant difference was found in their mutagenicity.

Identification of a novel allele HLA-DRB1*0114 in two Chinese marrow donors by sequence-based typing.

A novel human leukocyte antigen (HLA) class II, DRB1*0114, has been identified in routine typing of bone marrow donors for the Chinese Marrow Donor Registry in Shaanxi, China. The DRB1*0114 allele differs from DRB1*0101 by three nucleotide substitutions, causing two amino acid changes.

Hypoxanthine phosphoribosyl transferase assay of lead mutagenicity on keratinocytes.

An improved hypoxanthine phosphoribosyl transferase (HPRT) assay system was used to investigate the genotoxicity in human and rat keratinocytes exposed to Pb2+ 0.1-100 mumol.ml-1 in vitro. Autoradiography was applied to determine the number of labeled cells/cm2 of culture with [3H]TdR and liquid scintillation spectrometry was used to determine the incorporation of [3H]TdR into DNA counting of 6-thioguanine (TG)-resistant cells. The ratio between the number of labeled cells in the Pb2+ treated group (T) and in the control group (C) was calculated. When the cells exposed to Pb2+ 6 mumol.L-1 for 4 h, the T/C ratios reached 1.75 (scintillation, S), and 2.07 (autoradiography, A) in human and 1.37 (S), and 1.77 (A) in rat cells. A positive relation existed between the concentration of Pb2+ and mutagenicity. Lead should be considered as a weak mutagen in human and rat keratinocytes.

Toxic risks from inhalation of bacterial endotoxin.

A potential risk for workers exposed to inhalation of endotoxin, as well as the primary tissue changes, is the possibility of subsequent development of adult respiratory distress syndrome. Accordingly, hamsters were administered one hour aerosols of Enterobacter agglomerans and allowed to rest for six hours to produce maximum microlesions in the lung. One hour before this peak, the animals were injected intravascularly with the same suspension used in the aerosol. After one hour the animals were killed and bronchopulmonary lavages were made for analysis of free lung cells. As anaesthesia alone has been reported to be one of the operative procedures that enhance the effect of previous exposure to endotoxin, controls had to include sham anaesthesia with no intravascular injection. Endotoxin inhalation induced significant increases in total number of pulmonary leucocytes, mostly neutrophils, but with a concomitant relative decrease in number of alveolar macrophages. These polymorphonuclear neutrophils are not seen in morphometric analysis of the alveoli. Of greater interest was the large increase in erythrocytes suggesting pulmonary haemorrhages. Such changes were not due to intravenous bacterial alone, and were only significant when the bacteria were inhaled. Taken together with the other known inflammatory effects of inhaled bacteria or bacteria containing endotoxin, such inhalation in an occupational setting constitutes an increasingly recognised risk for workers.

Mutagenicity of scooter exhaust particulate matter.

By using the in vitro Ames Salmonella/microsomal assay and the in vivo mouse micronucleus assay, studies were performed to evaluate the genotoxicity of gasoline exhaust particulate matter generated from five different domestic and imported scooters. In the Ames assay, treatment of test strains TA98 and TA100 with solvent extracts of particulate matter from four of five scooter models caused an increase in the number of histidine-independent colonies over the background in TA98 without S9 mix. Positive results were also obtained from the micronucleus assay. The frequencies of bone marrow micronucleated polychromatic erythrocytes were significantly higher in the treated compared to the nontreated animals, and the increases in the frequencies were not significantly different among the five types of scooters. Analyses of chemical components showed that scooter exhaust particulate matter contained more than 100 different substances including polycyclic aromatic hydrocarbons.

Central nitric oxide donors attenuate cardiovascular and central norepinephrine responses to stress.

An earlier study showed that norepinephrine (NE) was released in the paraventricular nucleus (PVN) and posterior hypothalamus (PH) along with increases of mean arterial pressure (MAP) and heart rate (HR) during shaker stress (SS). Here we investigated the possibility that nitric oxide (NO) donors, infused into hypothalamus, could modulate responses to SS. In conscious rats, an injector-microdialysis probe, for direct application of donor and collection of extracellular NE, respectively, was inserted into PVN or PH; MAP and HR were recorded continuously from conscious rats. The NO donor, molsidomine (Mol), infused 5 or 30 min before SS, did not alter baseline values of NE, MAP, or HR, but did attenuate changes elicited by 5 min of SS; methylene blue blocked the effects of Mol. The NO donor, sodium nitroprusside, was much less effective than Mol as a modulator of stress-related effects. The results indicate that MAP, HR, and hypothalamic NE responses to environmental stress, but not baseline values, can be modulated by NO donors in the hypothalamus.

Mutagenicity of various organic fractions of diesel exhaust particles.

Treated with various organic fractions of Diesel Exhaust Particles (DEP), the Ames test withSalmonella typhimurium strains TA98 and TA100, and the mice micronucleus test were employed to study the mutagenic activity in the bacterial reverse mutation system, with and without a mammalian S(9) activation component, and the clastogenic activity in mice polychromatic erythrocyte (PCE) stem cells. Extracted ultrasonically with dichloromethane then using the acid and base separated reaction and column chromatography, DEP were divided into five organic fractions. They are the organic acid fraction (Fl), the organic base fraction (F2), the aliphatic hydrocarbon fraction (F3), the aromatic hydrocarbon fraction (F4) and the polar fraction (F5). Results showed that an increase in the counted numbers of histidine revertants on theSalmonella TA100 and TA98 was observed with or without (S(9) mix), but these activities were more pronounced in the TA98 strains especially in the absence of the S(9) mix. These results suggest that the organic fractions of DEP contain mainly compounds with direct frame-shift mutaganicity. Positive results were also obtained from mice micronucleus assay. The frequency of mice bone marrow micronucleated polychromatic erythrocytes (PCE) was increased using this assay and it showed a definite dose-response relationship. The results suggest that various organic fractions could affect spindle fiber function or formation in mammalian cells. Compared with the results of different organic fraction, the effects of the F2, F4 and F5 were found to be stronger than those of other fractions. Based on the findings obtaind in the Ames and micronucleus tests, DEPs have genotoxic effects in both of the test systems.