Multiple steady states with distinct cellular metabolism in continuous culture of mammalian cells.

Mammalian cells have the ability to proliferate under different nutrient environments by utilizing different combinations of the nutrients, especially glucose and the amino acids. Under the conditions often used in in vitro cultivation, the cells consume glucose and amino acids in great excess of what is needed for making up biomass and products. They also produce large amounts of metabolites with lactate, ammonia, and some non-essential amino acids such as alanine as the most dominant ones. By controlling glucose and glutamine at low levels, cellular metabolism can be altered and can result in reduced glucose and glutamine consumption as well as in reduced metabolite formation. Using a fed-batch reactor to manipulate glucose at a low level (as compared to a typical batch culture), cell metabolism was altered to a state with substantially reduced lactate production. The culture was then switched to a continuous mode and allowed to reach a steady-state. At this steady-state, the concentrations of cells and antibody were substantially higher than a control culture that was initiated from a batch culture without first altering cellular metabolism. The lactate and other metabolite concentrations were also substantially reduced as compared to the control culture. This newly observed steady-state was achieved at the same dilution rate and feed medium as the control culture. The paths leading to the two steady-states, however, were different. These results demonstrate steady-state multiplicity. At this new steady-state, not only was glucose metabolism altered, but the metabolism of amino acids was altered as well. The amino acid metabolism in the new steady-state was more balanced, and the excretion of non-essential amino acids and ammonia was substantially lower. This approach of reaching a more desirable steady-state with higher concentrations of cells and product opens a new avenue for high-density- and high-productivity-cell culture.

The characteristic study of TSP, PM2.5-10 and PM2.5 in the rural site of central Taiwan.

The total suspended particle (TSP), PM2.5-10 (aerodynamic diameter less than 10 microns) and PM2.5 concentration (aerodynamic diameter less than 2.5 microns) concentrations were sampled by PS-1 and Universal sampler on the roof (25 m) of the Medical and Engineering Building in the campus of Hungkuang Institute of Technology (HKIT) which is located at a height of 500 m on Da Du Mountain. The results indicated that average TSP, PM2.5-10 and PM2.5 concentrations are 0.42, 0.34 and 0.019 mg/m3 in the day time, respectively and are 0.32, 0.26 and 0.017 mg/m3 in the night time, respectively. The ratios of PM2.5-10/TSP were from 76% to 85% and from 50% to 91% for day and night period, respectively. It indicated that the major composition in the total suspended particles was PM2.5-10 in the rural site. The relationship between TSP and PM2.5-10 is TSP = 1.16PM2.5-10 + 0.027 and TSP = 1.01 PM2.5-10 + 0.058 in the day and night time, respectively. The correlation coefficient (R2) is 0.98 and 0.97 for day and night period, respectively. The relationship between PM2.5-10 and PM2.5 is PM2.5 = 0.0005PM2.5-10 + 0.019 and PM2.5 = 0.037PM2.5-10 + 0.0076 in the day and night period, respectively. The correlation coefficient (R2) is 3E-5 and 0.67 for day and night period, respectively. The relationships between TSP, PM2.5-10, PM2.5 particle concentrations and wind speed (R2) in the day time are 0.71, 0.64, 0.43, respectively and are 0.83, 0.79, 0.57, respectively in the night time. The proposed reasons are that there are more activities caused by people (students) and natural living animals which absorbed some of the particles during the day time. Thus, the correlation coefficients for the night time are better than those of day time. The particle size distributions are both bimodel in the day and night time. The major peaks in the day time appear in the particle diameter between 0.031-0.056 micron and 3.16-5.62 microns in the day period and appear between 0.017-0.031 micron and 1.78-3.16 microns in the night period. The results indicate that the particle size distribution in the day time tends to be of larger particle size mode than the night time.

Anomalous inferior and superior venae cavae with oculoauriculovertebral defect: review of Goldenhar complex and malformations of left-right asymmetry.

We observed a girl with an interrupted, left inferior vena cava with hemiazygous continuation, bilateral superior venae cavae, heart defects, and sacral agenesis. She had macrostomia and bilateral ear tags and pits, as in oculoauriculovertebral defect. Maternal diabetes was present. The combination, which we call OAV-heterotaxia complex, supports the view that some cases of oculoauriculovertebral defect may be part of a midline field defect of blastogenesis.

Muscle substrate utilization from alveolar gas exchange in trained cyclists.

The respiratory exchange ratio (R) during steady-state exercise is equivalent to whole-body respiratory quotient (RQ), but does not represent muscle metabolism alone. If steady-state values of carbon dioxide production (VCO2) and oxygen uptake (VO2) are plotted for different work rates, the slope of the line fitting these points should estimate muscle RQ. Twelve cyclists randomly performed five 8-min, constant work rate tests of 40, 80, 120, 160 and 200 W. Whole-body R, averaged over the final 2 min of each exercise bout, increased with increasing work rate. When VCO2 was plotted as a function of VO2, the regression lines through the five points displayed excellent linearity, had negative y-intercepts, and a slope of 0.915 (0.043) [mean (SD)], which was greater than the whole-body R at any individual work rate [range 0.793 (0.027) at 40 W to 0.875 (0.037) at 200 W]. This slope was comparable to the lower slope of the VCO2 versus VO2 plot of an increasing work rate (ramp) protocol [0.908 (0.054)]. We conclude that, during mild and moderate exercise of relatively short duration, contracting muscle has a high and constant RQ, indicating that carbohydrate is the predominant metabolic substrate. Whole-body R does not accurately reflect muscle substrate utilization and probably underestimates muscle RQ at a given work rate.

Modulation of experimental alcohol-induced liver disease by cytochrome P450 2E1 inhibitors.

This study was done to determine if a relationship exists between CYP2E1 induction by ethanol, lipid peroxidation, and liver pathology in experimental alcohol-induced liver disease in the rat. Rats were fed ethanol with or without diallyl sulfide (DAS) or phenethyl isothiocyanate (PIC) intragastrically for 1 month. CYP2E1 induction by ethanol was correlated with lipid peroxidation, liver microsomal CYP2E1 hydroxylation of paranitrophenol, and the liver pathology score using the data from the PIC-fed rats. Some of the data from the ethanol and DAS-fed rats were not included here because they have been reported elsewhere. Microsomal CYP2E1 protein levels induction by ethanol was decreased by PIC ingestion. Similarly, PIC reduced the increase microsomal reduced form of nicotinamide-adenine dinucleotide (NADPH)-dependent lipid peroxidation and p-nitrophenol hydroxylase (PNPH) activity, induced by ethanol feeding. The lipid peroxidation was reduced to below control levels; however, the pathology score was partially but not significantly reduced by isothiocyanate feeding. CYP2E1 messenger RNA (mRNA) was decreased by both inhibitors of CYP2E1. Immunohistochemical staining of liver for CYP2E1 protein showed that the lobular distribution of the isozyme changed from the centrilobular to a diffuse pattern, with an increase in the periportal region when the CYP2E1 inhibitors were fed with ethanol, and that this change correlated with the change in the distribution of fat in the lobule. The data support the idea that there is a link between CYP2E1 induction by ethanol and the early phase of ethanol-induced liver injury in this rat model. This link may involve lipid peroxidation, but other factors related to CYP2E1 induction must also be involved.

Tau protein in cerebrospinal fluid as an aid in the diagnosis of Alzheimer's disease.

Neurofibrillary tangles and dystrophic neurites are characteristic pathological features found in the brains of Alzheimer's disease (AD) patients. A major constituent of these lesions is the cytoskeletal protein tau. This study examined whether the measurement of tau in cerebral spinal fluid (CSF) has value in the diagnosis of AD. Seventy-seven subjects were enrolled in this prospective study: These included AD (N = 24), Neurological Controls (dementing diseases/syndromes, N = 26), Normal Controls (N = 14), and Others (N = 13). CSF was obtained by lumbar puncture, and tau concentrations (pg/mL) were determined using a dual monoclonal antibody microplate immunoassay. The mean tau value for AD subjects (1,430 +/- 739) was significantly different from Neurological Control subjects (790 +/- 579) (p < 0.001) and Normal Control subjects (816 +/- 355) (p < 0.001). Tau values were elevated in two Neurological Control subjects, one with Binswanger's disease (age 75) and one with depression (age 90). Tau values were also elevated in three Normal Control subjects; two were subjects with a family history of AD. Tau concentrations did not correlate significantly with age in AD subjects (r = 0.05, p = 0.82) or in Normal Control subjects (r = -0.49, p = 0.08). Tau also did not correlate with severity of cognitive impairment in AD subjects (r = -0.03, p = 0.91) or duration of AD symptoms (r = 0.16, p = 0.52). Based on these results and others, CSF levels of tau protein may provide a useful biochemical marker to aid in the clinical diagnosis of AD.

Role of cytochrome P4502E1 in alcoholic liver disease pathogenesis.

The intragastric tube feeding model is ideal for the study of the role of dietary factors and the effect of drugs on experimental alcoholic liver disease (ALD), since the model allows us to study the effect of a single variable in the diet on the pathology of liver where the blood alcohol level (BAL) is maintained over 150 mg%. By varying the dietary fatty acid composition we showed that the pathology was worsened by increasing linoleic acid or polyunsaturated fatty acids (PUFAs) in the diet where cytochrome P4502E1 (CYP2E1) was increased posttranslationally by high BAL. Concomitant with the increase in CYP2E1 there was evidence for an increase in lipid peroxidation (LP) by microsomes. Protein adducts of the products of LP were increased in the blood. Isoniazid (INH) enhanced this process and the pathology of ALD when INH was fed at therapeutic levels with ethanol. Preliminary studies show that diallyl sulfide, which inhibits and destroys liver CYP2E1 selectively, also modified the pathologic effects of ethanol. Thus we postulate that CYP2E1 induction plays a central role in the pathogenesis of ALD.

A clinically relevant canine lung cancer model.

Research on early human lung cancer is difficult; we have sought a canine correlate. Regimens included endobronchial submucosal injections and topical focal applications of benzo[a]pyrene, nitrosomethylurea, dimethylbenzanthracene, and methylcholanthrene, singly or in combinations. Sustained-release discs were placed into lung parenchyma or sutured into major bronchi. Tracheal segments were isolated as cervical pedicle grafts. Gross and histological evolution was reproducible. Columnar and basal hyperplasia and squamous metaplasia were early changes. Atypia occurred within 6 weeks and was found in all dogs within 16 to 18 weeks. Invasive cancers occurred within 8 to 65 months. No tracheal graft developed cancer. Of 15 dogs with parenchymal sustained-release implants, 1 to date has developed cancer in 8 months. Four endobronchial regimens have produced 16 cancers in 56 lungs at risk for 18 to 65 months. No cancers developed in 23 lungs at risk from eight other regimens. Of 10 dogs at risk for unilateral endobronchial cancer, 5 have had cancer. Of 23 dogs with both lungs at risk, 9 developed cancer. We have shown focal carcinogenesis with well-defined pathogenesis and an extended preneoplastic period at predictable sites in a lung cancer model.

Sustained release of benzo(a)pyrene from silicone polymer into the tracheobronchial tree of hamsters and dogs.

A method was developed to expose specific sites of the hamster and canine tracheobronchial tree to benzo(a)pyrene (BP) at quantitatively sustained rates. Implants for sustained release were formed by incorporating BP in a silicone rubber matrix at concentrations of 9.05 to 12%. Forty-nine hamsters and 12 dogs had a total of 86 implants surgically adhered to the tracheobronchial mucosa for up to 200 days. BP was released from the implants in hamsters and dogs as a first-order exponential function with a half-time of 54.8 and 44.5 days, respectively. Pathogenesis was progressively time and dose dependent. Squamous metaplasia with atypia regularly occurred in dogs within 150 days or after 7.17 mg BP and in hamsters after 50 days or 288 microgram BP. Bronchogenic cancers occurred in 93% of our hamsters after 100 days and 467 microgram BP. This method has applicability potentially as a bioassay for evaluating carcinogens in hamsters and currently as a means of producing a model of lung cancer in which neoplasia is induced at precise, selected sites.

Studies of ethylene-forming system in rat liver extract.

Evidence of enzymatic formation of ethylene from methionine by rat liver extracts is presented. The ethylene production is closely associated with growth of the animal. The conversion of L-methionine to ehtylene is oxygen dependent. Substrate analogue studies show that the ethylene-forming system is structurally specific and requires in the center of the molecule alpha-CH2-CH2- with one end attached to an unencumbered sulfur atom from a thioether moiety and the other end attached to a carboxyl group. Sylfhydryl agents are found to be very effective inhibitors of the ethylene-forming system. The finding of alpha-keto-4-methylthiobutyric acid to be a more efficient precursor of ethylene production suggests the possibility that alpha-keto-4-methylthiobutyric acid may be an intermediate in the biosynthesis of ethylene from methionine in mammalian tissues.

Catecholamine requirement for hamster sperm motility in vitro.

Homogenates of hamster and bovine glands contain a "sperm motility factor" (SMF) that stimulates the motility of hamster epididymal spermatozoa in vitro. The potency of these adrenal preparations was severely attenuated after gel filtration on a Sephadex G-10 column. This loss of activity was ascribed to the retardation and separation of co-factors for SMF which appeared to be catecholamines. The sperm motility-stimulating activity of the SMF-containing fractions was fully restored by addition of either the 'retarded' fractions or catecholamines (epinephrine or norepinephrine). Neither the catecholamines nor the 'retarded' fractions were able to sustain vigorous sperm motility in the absence of the SMF-containing fractions. The potentiating action of catecholamines on SMF was mimicked by the adrenergic agonists isoproterenol and phenylephrine and inhibited by the alpha-adrenergic antagonist phentolamine, but not by the beta-adrenergic antagonist propranolol. Our results indicate that one or more catecholamines are essential co-factors of SMF and demonstrate that hamster spermatozoa require catecholamines for their motility in vitro.

Persistent creatine kinase MB isoenzyme without cardiac disease.

Although the creatine kinase MB isoenzyme may be useful in the diagnosis of myocardial injury, it is also found in the sera of patients with a number of noncardiac diseases. It may also occur in apparently normal individuals, as described in this case report. Hence, the diagnosis of myocardial injury should never be based solely on the presence of creatine kinase MB isoenzyme in the serum, but should be supported by additional clinical and laboratory data.

An automated bichromatic measurement of serum immunoglobulins.

A new, rapid, discrete sampling and automated bichromatic procedure for the measurement of various soluble antigen-antibody complexes of immunoglobulins is described. The incorporation of polymeric substances and salts into the reaction mixture has greatly increased the sensitivity, shortened the reaction time and decreased the cost of the tests. Statistical analyses of data from this method have shown them to correlate well with values obtained by a radial immunodiffusion procedure. The automated procedure is simple and specific, it can be run at a rate of 90 specimens an hour.

Sustained-release implants of chemical carcinogens in the canine tracheobronchial tree.

A proved carcinogen, benzo[a]pyrene (BP), was incorporated into liquid silicone rubber polymer which was then vulcanized into solid form. Discs of BP polymer containing 9.05 to 12.12% BP were sutured into the trachea and right main bronchi of 10 dogs which were observed for 3 to 8 months. The discs were either in contact with the endobronchial surface of the mucosa or placed into submucosal pockets. All discs that were implanted submucosally remain in place. Only 2 discs implanted by the fixation method were extruded. The pocket implants of BP polymer caused ulceration and erosion of the mucosa; these changes did not occur from discs of polymer without carcinogen. To date these implants have induced squamous metaplasia with atypical changes. A method for sustained-release implantation of carcinogens in the canine tracheobronchial tree has been developed.