Cell suspensions

Exp Neurol, 1990 Jan, 107(1), 11 - 22
Synaptic connections formed by grafts of different types of cholinergic neurons in the host hippocampus; Clarke DJ et al.; The present experiment was performed to determine whether different types of grafted central cholinergic neurons are able to form synaptic contacts with host hippocampal neurons . Grafts from the septal-diagonal band area, which contain the neurons that normally innervate the hippocampal formation, were compared to those from the nucleus basalis magnocellularis region (NBM), the striatum, the pontomesencephalic tegmentum of the brain stem, and the spinal cord . The regions were dissected from 14- to 16-day-old rat fetuses, and the same number of viable cells (35 x 10(4} from each of the different regions was stereotaxically injected as a cell suspension into the hippocampus of rats subjected to a complete fimbria-fornix lesion, transecting the intrinsic septohippocampal pathways . At 14 to 17 weeks after transplantation, the brains were processed for choline acetyltransferase (ChAT) immunocytochemistry at the light and electron microscopic levels and acetylcholinesterase (AChE) histochemistry at the light microscopic level . There was a great variation in the number of surviving ChAT-positive cells among the different graft types . The septal grafts contained the highest number of ChAT-positive cells, and the striatal grafts showed the lowest numbers . The NBM, brain stem, and spinal cord grafts were in between . The differences in the number of ChAT-positive neurons between the groups matched, in general, the differences found in the magnitude of graft-derived AChE-positive fiber growth into the host hippocampal formation . At the electron microscopical level, all types of grafts were capable of forming synaptic contacts with host elements, however, with vast differences in the number of synapses found . The septal grafts produced the highest number of contacts, whereas the striatal and spinal cord grafts produced very few contacts . The ultrastructure of the cholinergic fibers from grafts obtained from the forebrain areas, i.e., septum, NBM, and striatum all appeared normal, whereas brain stem and spinal cord grafts produced different types of anomalies . The results show that grafted cholinergic neurons, that normally do not innervate the hippocampus, can send axons and form synaptic contacts in the host hippocampus . The ability to reinnervate the denervated hippocampal target appears to be shared by the embryologically closely related forebrain cholinergic neuron types, i.e., the septal, NBM, and striatal neurons . The marked differences in overall fiber ingrowth and number of synapses observed between these different types of grafts could be explained largely on the basis of differences in survivability of each grafted neuron type . By contrast, the reinnervation obtained from the grafted brain stem and spinal cord neurons were both quantitatively and qualitatively abnormal.(ABSTRACT TRUNCATED AT 400 WORDS)

J Clin Endocrinol Metab, 1990 Jan, 70(1), 28 - 34
Steroidogenesis in an estrogen-producing adrenal tumor in a young woman: comparison with steroid profiles associated with cortisol- and androgen-producing tumors; McKenna TJ et al.; There is only one previous report of an estrogen-secreting adrenal tumor occurring in a woman during reproductive years . Our patient presented with mild hirsutism associated with menstrual bleeding every 3-6 weeks . The occurrence of apparently intermenstrual bleeding prompted an evaluation of estrogen levels . Markedly elevated plasma estrone levels were found (860-2305 pmol/L; normal, 50-340) . Lesser relative elevations in 11-deoxycortisol and androstenedione were noted . Computed tomographic scanning of the adrenal glands identified a large tumor, which was subsequently resected . Estrone levels fell to 120 pmol/L, and all other abnormalities were corrected . Eighteen months after adrenalectomy, ovulation occurred regularly, and steroid levels were entirely normal . Steroid production in a cell suspension made from tissue obtained from the 190-g tumor was compared with that occurring in normal human adrenal cells . The production of estrone by the tumor cells was 40-fold greater than that by normal adrenal cells . There was also a mild excess of 11-deoxycortisol produced by tumor cells, but the tumor cells were less than 50% as efficient as normal cells in producing cortisol, dehydroepiandrosterone, androstenedione, testosterone, and dehydroepiandrosterone sulfate . Examination of the steroid profile in plasma occurring in three other patients with adrenal tumors reveals that while elevations in estrone occur frequently, this is usually due to the peripheral conversion of very high levels of androstenedione . Estrone, androstenedione, and 11-deoxycortisol plasma levels were elevated in all four patients; dehydroepiandrosterone sulfate was elevated in only two of four patients . After resection of one of these tumors, all steroid levels remained normal despite the occurrence of extensive metastases . These observations confirm the difficulty of making a diagnosis of estrogen excess in a woman during reproductive years because of the paucity of physical signs . The acquisition of aromatase activity was clearly demonstrated by tumor cells from our patient in vitro . Elevated plasma concentrations of estrone, androstenedione, and 11-deoxycortisol provide useful markers for adrenal tumors, but no one steroid can be relied upon in all tumors, and metastases may lack the steroidogenic capabilities of the primary tumor.

Exp Brain Res, 1990, 82(3), 641 - 50
Intrahippocampal cholinergic grafts in aged rats compensate impairments in a radial maze and in a place learning task; Schenk F et al.; Age-related cognitive impairments were studied in rats kept in semi-enriched conditions during their whole life, and tested during ontogeny and adult life in various classical spatial tasks . In addition, the effect of intrahippocampal grafts of fetal septal-diagonal band tissue, rich in cholinergic neurons, was studied in some of these subjects . The rats received bilateral cell suspensions when aged 23-24 months . Starting 4 weeks after grafting, they were trained during 5 weeks in an 8-arm maze made of connected plexiglass tunnels . No age-related impairment was detected during the first eight trials, when the maze shape was that of a classical radial maze in which the rats had already been trained when young . The older rats were impaired when the task was made more difficult by rendering two arms parallel to each other . They developed an important neglect of one of the parallel tunnels resulting in a high amount of errors before completion of the task . In addition, the old rats developed a systematic response pattern of visits to adjacent arms in a sequence, which was not observed in the younger subjects . None of these behaviours were observed in the old rats with a septal transplant . Sixteen weeks after grafting, another experiment was conducted in a homing hole board task . Rats were allowed to escape from a large circular arena through one hole out of many, and to reach home via a flexible tube under the table . The escape hole was at a fixed position according to distant room cues, and olfactory cues were made irrelevant by rotating the table between the trials . An additional cue was placed on the escape position . No age-related difference in escape was observed during training . During a probe trial with no hole connected and no proximal cue present, the old untreated rats were less clearly focussed on the training sector than were either the younger or the grafted old subjects . Taken together, these experiments indicate that enriched housing conditions and spatial training during adult life do not protect against all age-related deterioration in spatial ability . However, it might be that the considerable improvement observed in the grafted subjects results from an interaction between the graft treatment and the housing conditions.

Dev Neurosci, 1990, 12(4-5), 326 - 39
The myelin-deficient rat mutant: partial recovery of oligodendrocyte maturation in vitro; Espinosa de los Monteros A et al.; The morphological and immunocytochemical identification and characterization of the myelin-forming cell, the oligodendrocyte, have defined a model system for developmental studies . The myelin-deficient (md) rat mutant lacks myelin in the central nervous system and fails to express the normal developmental increase in oligodendroglial and myelin markers, apparently as a consequence of a point mutation in the proteolipid protein gene . In the present work, we compared the developmental pattern of primary glial cultures derived from newborn md rat brains to those derived from wild-type animals . Brain cell suspensions were prepared from each rat pup and cultured separately . We found by immunocytochemical and enzymatic analyses for the various markers that the developmental cascade of oligodendroglial marker expression is delayed, oligodendrocytes failing to mature compared to normal cultures . However, a partial recovery of marker expression was observed in md-derived cultures as compared to development previously reported in the intact md animals . We suggest that the partial recovery of the sequential expression of oligodendroglial markers may be due to a supportive environment provided to the oligodendrocyte progenitor cell (1) by tissue culture conditions or (2) by the absence of the blood-brain barrier in contrast to its presence in the intact animal.

Cancer Immunol Immunother, 1990, 32(3), 173 - 8
Postoperative active specific immunization in curatively resected colorectal cancer patients with a virus-modified autologous tumor cell vaccine; Lehner B et al.; Active specific immunotherapy was performed in a phase I study in 20 colorectal cancer patients after surgical resection of the tumor . An autologous tumor cell vaccine surface modified by Newcastle disease virus (NDV) was used, which showed the following characteristics . After mechanical and enzymatic dissociation of the tumor tissue an average of 5 x 10(7) cells/g tissue was obtained . According to trypan blue dye exclusion assay the average viability was 72% . Following irradiation (200 Gy) the inactivation of proliferative activity of the cells could be demonstrated by the absence of incorporation of 3H-labelled thymidine . The cells were, however, still metabolically active as shown by the incorporation of {3H}-uridine and a mixture of 3H-labelled amino acids . Epithelium-specific antigens (detected by mAb HEA125) were expressed on more than 75% cells of the cell suspension indicating a high amount of (epithelium-derived) tumor cells . In order to increase the immunogenicity of the tumor cells the suspended cells were infected by the nonlytic, apathogenic Ulster strain of NDV . The successful modification of tumor cells with NDV could be shown by electron microscopy . Three weeks postoperatively cells were thawed, virus-modified, and inoculated intradermally in the upper thigh . Several cell and virus concentrations were tested in each patient . As control, tumor cells without NDV, NDV alone and normal colon mucosa were used . The number of tumor cells ranged from 2 x 10(6) up to 2 x 10(7) cells and NDV concentrations from 4 to 64 hemagglutination units (HU) were tested . Sixteen patients responded with a delayed-type hypersensitivity (DTH) skin reaction to the vaccine . The best DTH reaction, measured 24 h following vaccination, was obtained using a vaccine consisting of 1 x 10(7) tumor cells and 32 HU NDV (median induration of 8 mm) . Response to NDV alone was seen in 2 patients only (median induration of 3 mm); 12 patients responded to tumor cells (1 x 10(7) alone (median induration of 4 mm) . Of 10 patients tested with normal colorectal mucosa, 4 responded with a median induration of 3.5 mm . DTH responses to the vaccine of 1 x 10(7) tumor cells and 32 HU NDV increased throughout the repeated vaccinations to a median induration of 9.5 mm at the end of the therapy . No severe side-effects in the course of the immunotherapy, except for mild fever in 4/20 patients, were observed . The results of our phase I study show that this type of autologous colorectal tumor cell vaccine is ready for a large clinical trial to prove its efficacy.

Yao Xue Xue Bao, 1990, 25(6), 469 - 72
{The effects of HCG and LHRH-A on progesterone secretion by human placenta villi cells of early and term gestation in vitro}; Zhu BT et al.; In the present study, the method of collagenase-pepsin prepared cell suspension of human placental villi from early (10-12 weeks) and term (38-40 weeks) gestation cultured in vitro combined with progesterone radioimmunoassay was employed . The results indicated that: 1 . In the placenta villi cells from early and term gestation in vitro, both exogenous and endogenous hCG showed a stimulatory effect on progesterone secretion, and the effect was approximately proportional to the concentration of the exogenous hCG added . 2 . In the placenta villi cells from term gestation in vitro, after addition of sufficient rabbit antihCG IgG to the culture medium, LHRH-A exhibited no obvious influence on this hCG-independent progesterone secretion . However, in the absence of rabbit anti-hCG IgG in the culture medium, LHRH-A showed a stimulatory action on progesterone secretion probably via its stimulatory effect on hCG secretion which could subsequently increase the progesterone secretion . 3 . In the placenta villi cells from early gestation, LHRH-A exhibited an inhibitory effect on both hCG-independent and hCG-dependent progesterone secretion.

Clin Lab Haematol, 1990, 12 Suppl 1, 13 - 21
Recommended methods for the assignment of assay values to stabilized cell suspensions; England JM; Stabilized blood suspensions are required for the transfer of data from accurately calibrated reference instruments to service laboratory instruments . However, the more the cells are stabilized to increase shelf life, the less like fresh blood they become . Fixation is quite satisfactory for producing red count standards but it affects the flexibility, shape factor and the assigned MCV of the erythrocytes . Because of these problems values must be assigned indirectly by assigning values to fresh blood by reference methods and subsequently comparing fresh bloods with stabilized suspensions on a range of user instruments . The details for these methods are given and the reasons for these requirements are discussed.

Arch Dermatol Res, 1990, 282(6), 402 - 7
Electron microscopic study of cultured cells from the murine hair tissues: cell growth and differentiation; Tanigaki N et al.; The cultured hair cells from 4-day-old C3H mice were studied by electron microscopy . The hair roots isolated from the skin by collagenase digestion were dispersed into a cell suspension by treatment with a mixture of trypsin and ethylenediaminetetraacetate . The cells were cultured in MCDB-153 (a medium containing seven growth factors) for 1, 3, 6 or 13 days . The number of cultured cells on day 3 was twice that on day 1, and stayed at the same level until day 13 . By electron microscopy, some of the cells cultured for 1 day were seen to be undifferentiated and others already showed differentiation into various hair structures . Such differentiated cells disappeared on day 3 and most of the cells cultured for 3 days were undifferentiated . Cell cultured for 6 days were differentiated showing inner root sheath cell, hair cortical cell and medulla cell structures . The characteristics of these cultured cells corresponded well to those of in vivo cells of the hair tissues from the back skin of 7-day-old C3H mice . On day 13 degeneration occurred in the cultured cells . In none of these cultures were mesenchymal cells, such as fibroblasts, found . The present electron microscopic study reveals that immature cells obtained from mouse hair tissues proliferate in vitro and differentiate into several subpopulations corresponding to those of in vivo cell layers of hair tissues . The present culture technique may be useful for studies of hair cell growth and differentiation.

Tsitologiia, 1990, 32(7), 712 - 9
{The relationship of damage to and death of ascitic tumor cells during starvation to the ATP and free calcium content in the cells}; Gabai VL et al.; Ascite tumor cells EL-4 were incubated in conditions of energy starvation (Hanks salt solution with rothenone and without glucose) at 37 degrees C for 3 hours . Under these conditions, some structural cell damages appeared within the first hours: enlarging and flattening of the cells, blebbing, vacuolization of the cytoplasm, nuclear chromatin condensation . Later on, a share of cells with obvious damage decreased, whereas that of the cells stained with trypan blue (dead cells) much increased (up to 90% after a 3 hour incubation) . The cellular ATP decreased abruptly (up to 10% of the control) during the first 10 minutes of starvation . Free Ca2+ concentration increased within 1 hour of incubation more than two-fold . The conditions promoting Ca2+ influx (ionophore A23187 + Ca2+ in medium) accelerated the damage and cell death . However, the increase in free Ca2+ concentration did not trigger any damage in the energy-starved cells, since in the Ca2(+)-depleted medium (no increase in free Ca2(+)-concentration) the development of damages was not prevented . The damage initiation was irreversible: the addition of glucose to cell suspensions after 0.5-1 hour of their incubation in energy-starved condition did not prevent the development of damage, while ATP content in these cells was much increased.

Ann N Y Acad Sci, 1990, 600, 384 - 402; discussion 402-4
Aging and regenerative capacity of the rat serotonergic system . A morphological, neurochemical and behavioral analysis after transplantation of fetal raphe cells; Steinbusch HW et al.; Morphological dissimilarities between the brains of young (3 months) and aged (28 months and older) rats were demonstrated using serotonin-immunocytochemistry . A degeneration of the serotonergic system, noted as a decreased innervation and the appearance of enlarged or swollen varicosities, was observed particularly in the frontoparietal cortex, and the neostriatum of the aged rat brain . No direct relationship between this aberrant morphology and decrease in density of serotonin-innervation was found as we demonstrated a decline in fiber density without the appearance of aberrant serotonergic fibers in the hippocampus . HPLC analysis revealed that serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) levels in the frontoparietal cortex, hippocampus and raphe area were increased in the aged rat, while the 5-HT level in the caudate-putamen complex was not different from the young adult rat . The ratio 5-HIAA/5-HT, indicative of 5-HT turnover, appeared increased in the frontoparietal cortex, sensoric part, the caudate-putamen and the raphe area, while this ration in the frontoparietal cortex, motoric part and the hippocampus was not altered in the aged rat . Behavioral screening revealed a decrease spatial performance of aged males in a Morris Water-Maze task . To investigate whether the age of the host recipient was of influence on the regenerative capacity, a fetal raphe cell suspension of embryonic day E 15 was implanted in the caudate-putamen of young adult as well as aged rats . Neither differences in survival of the serotonergic cells nor in fiber outgrowth between both groups appeared five weeks after transplantation . Subsequently, transplantation of raphe cells in the hippocampus of young adult rats, after lesioning the hippocampal serotonergic innervation with 5,7-DHT, was performed to compare behavioral, morphological and neurochemical effects of the implants . It appeared that 11 months after transplantation the serotonergic innervation of the previously denervated hippocampus was greatly restored . There was a striking resemblance between the immunohistochemical and neurochemical data with respect to the increase in the amount of newly formed serotonergic fibers, the increase in uptake of {3H}-5-HT and in 5-HT and 5-HIAA levels . Also the behavior of lesioned and lesioned + transplanted males was rather similar to controls . In the behavioral tests we were mainly interested in hippocampal functioning, therefore orientation was of our prime interest . The other behavioral tests were only to confirm that the possible changes were linked to hypothalamic or extra-hypothalamic functions.(ABSTRACT TRUNCATED AT 400 WORDS)

Am J Pediatr Hematol Oncol, 1990 Fall, 12(3), 245 - 56
Ex vivo chemopurging of autologous bone marrow with 4-hydroperoxycyclophosphamide to eliminate occult leukemic cells . Laboratory and clinical observations; Yeager AM et al.; The application of autologous bone marrow transplantation (ABMT) in treating acute leukemias in children has been limited by the presence of residual occult viable leukemic cells in the marrow cell suspension . One approach to this problem is the ex vivo treatment ("purging") of the autograft to eradicate these tumor cells yet spare the normal lymphohematopoietic stem cells . Initial studies of acute myeloid leukemia (AML) in a rodent model demonstrated that incubation with 4-hydroperoxycyclophosphamide (4HC), a congener of cyclophosphamide and an active alkylating agent in aqueous solution, could effectively eliminate viable AML cells from marrow cell suspensions without apparent toxicity to normal stem cells . We have conducted clinical trials of ABMT with 4HC-treated marrow in children with acute leukemia in remission; marrow was collected, treated ex vivo with 4HC (100 micrograms/ml), and cryopreserved in liquid nitrogen until reinfusion . Children received pre-ABMT conditioning with either high-dose cyclophosphamide and total body irradiation (CY-TBI) for acute lymphocytic leukemia (ALL) or high-dose busulfan and cyclophosphamide (BU-CY) for AML . Of nine children who underwent ABMT with 4HC-treated marrow for ALL in second complete remission (CR2), all relapsed (eight in the marrow, one in the central nervous system) at a median of 5 months (range, 2-17) after ABMT and all have died with relapsed ALL or as a consequence of its treatment . Twenty-nine children with AML (five in CR1, 24 in CR2) received autografts with chemopurged marrow at a median remission duration of 3 months (range, 2-15) . Three patients died from sepsis during aplasia; 10 children (one in CR1 and nine in CR2) relapsed with AML at a median of 7 months (range, 2-23) after ABMT, for an actuarial relapse rate of 47% . Sixteen patients with AML (four in CR1, 12 in CR2) are in unmaintained remission at a median of 16 months (range, 6-102) after ABMT, for an actuarial disease-free survival of 49% . Although ABMT with 4HC-treated marrow appears to have a limited role in the treatment of children with ALL who lack a suitable related donor, the results in AML are encouraging and compare favorably with both syngeneic and allogeneic BMT in similar groups of patients.

Invasion Metastasis, 1990, 10(5), 281 - 8
Lymphatic metastases from the peritoneal cavity are increased in the postinflammatory state; Levine S et al.; Cell suspensions of chemically induced tumors (rhabdomyosarcoma) were transplanted into the peritoneal cavities of Lewis rats . In normal animals, the greater omentum was the main site of tumor growth, and transdiaphragmatic metastases to regional lymph nodes in the mediastinum were few and small . In animals during the healing phase of a chemical peritonitis, the greater omentum was fibrotic, shrunken, and inactivated . The loss of the scavenging function of the omentum was associated with wide dissemination of the tumor in the peritoneal cavity and increased access of the tumor to the lymphatic stomata on the peritoneal surface of the diaphragm . Number and size of transdiaphragmatic metastases in draining lymph nodes were greatly increased in this postinflammatory state.

Int J Syst Bacteriol, 1990 Jan, 40(1), 66 - 70
Arylsulfatase activity of Mycobacterium avium, M . intracellulare, and M . scrofulaceum; Falkinham JO 3rd; A rapid (3-h) arylsulfatase assay for cell suspensions of mycobacteria, in which p-nitrophenyl sulfate is used as the substrate, was developed . Arylsulfatase activity was found in cell suspensions of representative strains of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum grown without the substrate in either Middlebrook 7H9 medium containing 0.2% (wt/vol) glucose and 0.05% (vol/vol) Tween 80 or Dubos broth medium, but was absent in cells grown in a low-pH, minimal medium containing 1% (vol/vol) Tween 80 as the sole carbon source . The levels of arylsulfatase activity of representatives of all three species were equal whether the activity was measured at pH 5.5, 6.5, or 7.5 and whether the cells were suspended in phosphate or Tris buffer . The addition of high levels of sulfate (present in the low-pH, Tween 80-containing medium) to Middlebrook 7H9 medium resulted in significantly lower levels of arylsulfatase activity in strains of M . scrofulaceum, but did not affect the levels in either M . avium or M . intracellulare . The levels of arylsulfatase activity were highest in M . avium, intermediate in M . intracellulare, and lowest in M . scrofulaceum strains . Polyacrylamide gel electrophoresis of crude extracts from late-log-phase cells of representatives of each species produced activity bands of unique mobility (one in M . avium, three in M . intracellulare {82, 5, and 13%}, and two in M . scrofulaceum {60 and 40%}).

Acta Leprol, 1990, 7(2), 157 - 61
Isolation and characterization of infiltrates in the nerves of patients with neuritic leprosy; Kumar V et al.; A study was done on the characteristics of infiltrating cells in the nerves of 9 patients with pure neuritic leprosy, by preparing a single cell suspension . The patients had no skin lesion . Histopathological examination revealed that 2 of the 9 nerves showed granulomas characteristics of tuberculoid leprosy, while the remaining 7 had features of lepromatous granulomas . In the nerves showing tuberculoid granulomas, a high proportion of lymphocytes were T cells as they formed rosettes with sheep erythrocytes and only a few percent were EAC rosette forming cells . On the other hand, the nerves showing lepromatous granulomas contained only occasional lymphocytes which formed E and EAC rosettes . Macrophages from the granulomas of all the nerves were esterase positive, peroxidase negative, contained M . leprae and did not exhibit C3 surface receptors.

J Clin Gastroenterol, 1990, 12 Suppl 1, S177 - 80
Protective effect of sofalcone and 16,16-dimethyl-PGE2 on isolated rat gastric cells; Kobayashi K et al.; The effects of 16,16-dimethylprostaglandin E2 (dm-PGE2) and sofalcone on damage caused by ethanol in surface epithelial cells isolated from rat stomach were examined . The surface epithelial cells (SECs) accounted for 83% of the isolated gastric mucosal cells, mucus neck cells for 5%, parietal cells for 9%, and chief cells for 3% . To suspensions of SEC, either dm-PGE2 at concentrations of 10(-7), 10(-6), or 10(-5) M or sofalcone at concentrations from 5 X 10(-6) to 10(-4) M was added; some cells were treated with neither drug . Ten minutes later, ethanol was added to each cell suspension to a final concentration of 15%, and 5 min later the viability was evaluated by trypan blue exclusion . At 10(-6) M, dm-PGE2 reduced ethanol-induced cell damage most strongly (p less than 0.001) . Sofalcone also helped to prevent cell damage caused by ethanol in a dose-dependent manner . These results suggest that dm-PGE2 and sofalcone protect gastric mucosa not only from gross visible damage but they directly protect gastric cells from damage.

Folia Med Cracov, 1990, 31(1-2), 73 - 9
{Binding of various polycyclic aromatic hydrocarbons (PAH) by plasma albumin and blood cells}; Lipniak M et al.; We have studied in vivo and in vitro partition of pyrene, fluoranthene and benz(a)anthracene (BaA) between blood cells and plasma or phosphate buffer pH 7.4 and their binding to bovine serum albumin . The level of PAH in blood and plasma was determined in rats after i.v . administration of pyrene, fluoranthene and BaA in dose 20 mg/kg by gas chromatography method . In vitro 20 micrograms of PAH was added to blood samples or blood cell suspension . These samples were incubated at 37 degrees C . After 1 hr the concentration of PAH in blood cell and plasma or buffer was determined . The data indicate that whole pyrene and fluoranthene are distributed to blood cells . 39% of BaA in whole blood is present in blood cells and 56.9% in plasma . Plasma protein binding of PAH were determined by gel filtration 0.4 cm3 of PAH-protein solution was given on a Sephadex column . PAH binding parameters were obtained from simple linear regression of Scatchard plots of the data . In a albumin solution (0.4%) the number of binding sites was 2 for BaA, 3 for pyrene and 11 for fluoranthene . The binding constants for BaA, fluoranthene and pyrene were 0.17; 0.71 and 1.11 mol-1 X 10(4), respectively.

Pathology, 1990 Jan, 22(1), 5 - 9
Comparison of flow cytometry and retrospectively applied static cytometry on lymphoid tissue; Jones A et al.; Twenty cases of non-Hodgkin's malignant lymphoma were examined using both flow cytometry (FCM) and static cytometry (SCM) DNA analysis to detect aneuploidic cell populations . FCM was performed on fresh cell suspensions whilst SCM was performed retrospectively on formalin-fixed, paraffin-embedded samples of the same tissue . A total of 34 aneuploidic cell lines were detected by FCM compared to a total of 41 detected by SCM . Of the 20 cases 14 showed DNA indexes within 10% of each other for each method, indicating a reasonable degree of comparability between the two methods . Of the 6 cases which did not show comparable DNA indexes, 2 displayed similar cell lines for both methods, but the major abnormal cell populations were of different ploidy by SCM as compared to FCM . A qualitative comparison is thus possible between these two cases . The results suggest that retrospectively applied SCM ploidy analysis of lymphoid tissue is comparable to FCM on fresh tissue samples and can thus be used in retrospective studies of both prognostic and diagnostic significance.

Biorheology, 1990, 27(1), 47 - 65
Further investigations of red cell deformability with nickel mesh; Arai K et al.; Although the filtration method has been widely employed in red cell deformability studies, the structural irregularity of the pores of a Nuclepore polycarbonate membrane has always been a major problem . Anegawa, T . et al . (Clin . Hemorheol., 7, 1987) obtained a higher reproducibility with the filtration method using a newly designed thin metal film with pores engraved by the photofabrication technique . We further studied the pressure - flow rate relationship of red cell suspension employing this nickel mesh . The filtration of red cell suspensions through the nickel mesh was not influenced by leukocytes contamination or added leukocytes up to a leukocyte count of 250 cells/mm3 within an experimental limitation . On the other hand, the flow was greatly influenced by leukocytes contamination when the polycarbonate membrane was used . The nickel mesh was found to be useful in detecting major determinants of red cell deformability, such as cell geometry and internal cellular viscosity, and in detecting abnormalities of red cell deformability in a patient with microangiopathic hemolytic anemia . In conclusion, the present study clearly shows that the nickel mesh is preferable for investigating red cell deformability to the polycarbonate membrane from a quantitative point of view . This material should contribute to the physiologic and clinical investigation of red cell deformability.

Haematologica, 1990 Jan-Feb, 75 Suppl 1, 1 - 5
Processing and manipulation of human hemopoietic stem cells . The Italian Cooperative Study Group on Cell Manipulation in Hematology; Iacone A; In this paper, we reviewed the basic principles and methods of collection, processing and manipulation of marrow and peripheral haemopoietic progenitor cells . The advances in cell separation are due to the progress in blood bank technology and to the introduction of more sophisticated devices . This allows to process larger amounts of blood or marrow and to obtain a final single cell suspension . For this purpose, several procedures have been employed, including the unit gravity sedimentation, the centrifugal elutriation, the isopyknic sedimentation and the positive selection on columns of CD34+ cells . As expected, the emerging problems are the extreme heterogeneity of the methods employed and the unreliability of the quality control . The proposal for standardized protocols for each step of the various procedures is urgent, and the institution of multicenter studies is recommended.

Cancer Chemother Pharmacol, 1990, 26(2), 122 - 6
Hepatic tissue distribution of doxorubicin-loaded nanoparticles after i.v . administration in reticulosarcoma M 5076 metastasis-bearing mice; Chiannilkulchai N et al.; In our previous studies, doxorubicin-loaded polyisohexylcyanoacrylate nanoparticles have been proven to increase dramatically the antitumoral activity of the cytotoxic agent in metastasis-bearing mice . The experimental model consisted of metastases induced by i.v . inoculation of reticulosarcoma M 5076 cell suspension to C57BL/6 mice . The improved efficacy of the drug was noted in terms of either metastasis count or survival . Therefore, tissue-distribution studies of this drug delivery system within the metastatic liver after i.v . administration were undertaken to gain more insight into the mechanism of action . Doxorubicin measurements in healthy hepatic or neoplastic tissue were carried out together with histological examinations using transmission electron microscopy . These results demonstrated the hepatic tissue to be an efficient reservoir of the drug when it was injected associated with nanoparticles . Accumulation of biodegradable nanoparticles with associated doxorubicin in Kupffer cells created a gradient of drug concentration for a massive and prolonged diffusion of the free drug towards the neoplastic tissue.

Annu Rev Neurosci, 1990, 13, 415 - 40
Dopamine cell replacement: Parkinson's disease; Yurek DM et al.; Significant progress in neural transplantation has been observed over the last several decades . As a neuroanatomical tool, neural transplantation studies are able to examine the mechanisms involved in the development and integration of neurons into the complex neural circuitries of the brain . Today, embryonic neural tissue can be successfully transplanted as solid tissue chunks or as dissociated cell suspensions . Within the parenchyma of the brain, transplanted embryonic neurons develop mature morphology and do not appear to invoke an immunological response by the lost immune system . Not only do these neurons exhibit robust development but there is also evidence that transplanted neurons restore some degree of function to neurologically damaged circuitry; however, the extent of reintegration into the host neural circuitry still remains unclear . Moreover, the long-term survival and functioning of transplanted nerve cells also remains an unanswered question . Advances in the emerging field of genetic engineering may eventually lead to genetically modified neurons that are capable of synthesizing neurotrophic factors or missing neurotransmitters and restoring function in brain-damaged areas . The use of neural transplantation to replace damaged nerve cells in neurodegenerative disorders, such as Alzheimer's or Parkinson's disease, is promising based on our current knowledge . However, our basic scientific knowledge of neural transplants is incomplete and warrants a prudent approach toward application of neural transplantation techniques in clinical research.

Acta Obstet Gynecol Scand, 1990, 69(2), 153 - 9
Detection of human papilloma virus in women referred for colposcopy . A comparison between different diagnostic methods; Boden E et al.; Various methods presently available for the diagnosis of genital Human Papilloma Virus (HPV) were compared regarding their sensitivity in women referred for specific diagnosis and treatment because of atypical Pap smears or clinically suspected neoplasia . Colposcopic examination was performed in all cases . In addition to taking a second Pap smear, cell suspensions were made from 105 women and analysed by the Filter In Situ Hybridization (FISH) technique and tested for HPV 6 + 11 and HPV 16 + 18 + 31 . The FISH technique was also used for the possible detection of HPV-DNA in a reference material comprising 119 apparently healthy women with normal Pap smears . Colposcopically directed cervical biopsies in altogether 196 specimens were obtained from 155 women for histopathological examination and also for the detection of HPV-DNA by the Southern blot hybridization technique . These specimens were tested for the presence of HPV 6, 11, 16, 18, 31 and 33 . Three per cent of the women with and 34% without cytological signs of HPV in Pap smears had cervical intraepithelial neoplasia (CIN) III according to histopatology . CIN III was present in 35% of biopsies with and 59% of biopsies without histological signs of HPV in the biopsies . Altogether 46% of the women were HPV-DNA positive . Of the women analysed by Southern blot, 39% were HPV-DNA positive . Of the samples analysed by FISH, 27% with atypical cells were HPV-DNA positive, compared with 11% of the samples from reference women with normal cytology.(ABSTRACT TRUNCATED AT 250 WORDS)

Zh Nevropatol Psikhiatr Im S S Korsakova, 1990, 90(2), 12 - 5
{Prognostic value of NA K ATPase activity in multiple sclerosis}; Ierusalimskii AP et al.; The paper is concerned with the results of measuring the activity of Na+, K+, ATPase in red blood cell suspension in patients with different courses of multiple sclerosis (MS) within the range of physiological temperatures (34 to 42 degrees C) . Account was taken of the interrelation of structural phasic transitions in cell membranes to the changes in enzymatic activity . There was a spasmodic increase of the activity of Na+, K+, ATPase at 35 degrees C and 40 degrees C at the moment of the clinical signs of disease exacerbation together with a twofold lowering of the enzymatic activity at a temperature of 35 degrees C during remission . The rise of the enzymatic activity at a temperature of 35 degrees C anticipates clinical exacerbation and thus may be of prognostic importance in the determination of the type of the disease course.

J Steroid Biochem, 1990 Jan, 35(1), 127 - 32
Pituitary and gonadal function during physical exercise in the male rat; Harkonen M et al.; The effects of training and acute exercise on serum testosterone, luteinizing hormone (LH) and corticosterone levels and on testicular endocrine function in male rats were studied . In the first part of the study, the rats were trained progressively on a treadmill, over 8 weeks . Training did not change the basal levels of serum testosterone, LH and corticosterone, or the testicular concentrations of testosterone and its precursors progesterone and androstenedione . The levels of testicular LH (30.3 +/- 2.6 ng/g wet wt, mean +/- SEM) and lactogen (150 +/- 14 pg/g) receptors were unchanged after training . However, the capacity of testicular interstitial cell suspensions to produce cAMP and testosterone increased by 20-30% during in vitro gonadotropin stimulation . In the second part, the trained and untrained control animals underwent acute exhaustive exercise . Serum testosterone levels decreased by 74 and 42% in trained and untrained rats, respectively (P less than 0.02), and corticosterone rose by 182% in trained and 146% in untrained rats (P less than 0.01), whereas the LH level was unchanged . Testicular levels of testosterone and its precursors decreased, with the exception of unchanged androstenedione, in trained rats; the cAMP concentration was unchanged . In both trained and untrained rats, acute exercise decreased the capacity of interstitial cell suspensions to produce cAMP, whereas there were no consistent effects on testosterone production . Acute exercise had no effect on LH or lactogen receptors in testis tissue . In conclusion, training had no effect on serum or testicular androgen concentrations, but increased Leydig cell capacity to produce testosterone and cAMP . Acute exercise decreased serum and testicular testosterone concentrations without affecting serum LH . A direct inhibitory effect of the increased serum corticosterone level on the hypothalamic-pituitary level and/or testis may be the explanation for this finding.

Am J Physiol, 1990 Jan, 258(1 Pt 2), F52 - 60
Influence of Na+ intake on dopamine-induced inhibition of renal cortical Na(+)-K(+)-ATPase; Seri I et al.; The enzyme L-amino acid decarboxylase (L-AADC), found in abundance in rat proximal tubule cell cytosol, converts L-dopa to dopamine . Dopamine, in turn, suppresses proximal tubule sodium transport by inhibiting Na(+)-K(+)-ATPase activity . We sought to determine whether changes in dietary sodium intake in rats lead to adaptation of dopamine formation and dopamine-induced Na(+)-K(+)-ATPase inhibition . In rats on a high-salt (HS) diet, the maximal velocity (Vmax) of renal cortical L-AADC was 78 +/- 19% higher than that in rats on a low-salt (LS) diet . The Michaelis constant (Km) of the enzyme remained unchanged . In renal cortical tubule cell suspensions the L-dopa-induced inhibition of ouabain-sensitive oxygen consumption (QO2) was significantly greater in rats on HS diet than in rats on LS diet . Furthermore, L-dopa completely inhibited the nystatin-induced rise in QO2 in the HS but not in the LS group . Carbidopa, an inhibitor of L-AADC, abolished the L-dopa-induced inhibition of nystatin-stimulated QO2 in cells from HS rats and was without significant effect in cells from LS rats . L-Dopa-stimulated K+ efflux was greater in cells from HS rats at 28 +/- 1 nmol.min-1.mg protein-1, compared with 7 +/- 6 nmol.min-1.ng protein-1 in cells from LS rats . By contrast, ouabain-stimulated K+ efflux did not differ between the groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Br J Dermatol, 1990 Jan, 122(1), 33 - 42
Antigen presentation in patients with recrudescent orofacial herpes simplex virus infections; Vestey JP et al.; Recovery from epidermal herpes simplex virus (HSV) infection depends primarily on development of an effective cell-mediated immune response, possibly generated following antigen (Ag) presentation by epidermal cells (EC) . The ability of EC to present HSV Ag was investigated in 12 subjects with occasional recrudescent facial HSV infections . All had circulating HSV specific antibodies and cell-mediated immunity to the virus . Peripheral blood mononuclear cell suspensions, depleted of antigen presenting cells (APC) by glass adherence and then enriched for T cells by adsorption on nylon wool columns, did not proliferate in response to HSV Ag . Both EC suspensions, prepared from suction blister roofs, and glass-adherent peripheral blood mononuclear cells (AC) preincubated with ultraviolet-inactivated HSV, reconstituted the T-cell proliferative response to HSV . EC were more efficient than AC at presenting HSV Ag to T cells . Depletion of CD1+ cells from EC suspensions by cell sorting reduced their ability to present HSV Ag and augmentation of CD1+ cell numbers supplemented it . Preincubation of EC or AC with monoclonal antibodies to major histocompatibility complex class II antigens DP, DQ or DR, blocked the lymphoproliferative response to HSV Ag . Evidence was obtained that cells co-ordinately expressing products of the DP, DQ and DR loci are involved in presentation of HSV Ag by both EC and AC.

Arch Biochem Biophys, 1990 Jan, 276(1), 160 - 71
Stimulation of human erythrocyte 2,3-bisphosphoglycerate phosphatase by vanadate; Mendz GL et al.; The rates of vanadate-stimulated hydrolysis of 2,3-bisphosphoglycerate in metabolically competent erythrocytes and in hemolysates were determined from data on time courses up to 35 min employing 31P nuclear magnetic resonance spectroscopy . The enhanced rate of hydrolysis of the bisphosphate was attributed principally to the activation of the phosphatase activity of 2,3-bisphosphoglycerate synthase both in cell suspensions and in hemolysates . Information on the concentrations of vanadate and vanadyl present in the preparations was obtained employing 51V nuclear magnetic resonance spectroscopy and electron paramagnetic resonance spectroscopy . Redox reactions involving vanadium ions appeared to be important in establishing the final equilibrium concentrations of the oxy- and oxo-ions (vanadate and vanadyl, respectively), but the data suggested that the activation of the enzyme resulted from direct action of the vanadium ions on the enzyme and not as a consequence of the alteration in the equilibrium of intracellular oxidants and reductants.

Membr Biochem, 1990 Jan-Mar, 9(1), 47 - 60
An Escherichia coli mutant conditionally altered in respiratory chain components; Cox JC et al.; Nitrosoguanidine mutagenesis was employed to isolate an Escherichia coli mutant conditionally altered in respiratory chain components . Mutant R25 was able to grow on glucose, fructose, and glycerol but failed to grow on succinate and acetate (suc-) . Also, R25 exhibited leaky growth on DL-lactate, fumarate, and malate (lct*) . The lct* mutation pleiotropically affected a number of respiratory chain components and its expression was conditional with the growth substrate . Glucose-grown R25 resting cell suspensions oxidized DL-lactate and formate; however, these two substrates were not oxidized by fructose- or glycerol-grown cell suspensions . The same conditional pattern was observed for the concentration of cytochrome components, the membrane-associated oxidation of NADH and formate, and formate phenazine methosulfate (PMS) reductase activity; succinate oxidase and PMS reductase activities were not exhibited by membranes under any growth condition due to the suc- mutation . R25 membrane-associated H(+)-translocating ATPase activity was not conditional with the growth substrate . R25PC, a spontaneous lct+ suc- partial revertant of R25, did not exhibit the conditional pattern of R25 . The lct* mutation was found to map in the 27-30-min region and the suc- mutation in the 15-17-min region of the E . coli genome . Two distinct classes of R25 P1kc transductants were isolated that differed in both their growth response on succinate and DL-lactate and their oxidase activities.

Am J Med Genet Suppl, 1990, 7, 219 - 24
Intrathymic deficient expansion of T cell precursors in Down syndrome; Musiani P et al.; To correlate the intrinsic cellular immunodeficiency, which is a major cause of increased susceptibility to polytopic infections in Down syndrome (DS) patients, with the histologic abnormalities observed in the thymus of these patients, we have studied thymus fragments and thymocyte cell suspensions from 15 non-institutionalized DS subjects . Comparing to the control age-matched samples, a reduced thymic cortex and a distinct depletion of CD1-positive (+) cells was observed by immuno-histologic examination . The phenotypic analysis of unselected thymocytes showed a significant reduction of CD3+, CD1+, CD4+, and CD8+ cells . When the total thymocyte population was separated into 10 fractions, using a continuous Percoll density gradient, a difference in cell distribution was observed . DS thymuses are almost devoid of high-density thymocytes (fractions 6-9) while more than 75% of the cells were recovered in the lightest 3 fractions (Frs) . In addition, these thymuses were characterized by a marked depletion of CD1+ cells and by a conspicuous reduction of CD3+ cells normally present in the high-density Frs . On the other hand, the lightest 3 Frs of DS subjects were enriched in low-density CD1+ cells . Although enriched in these cells, normally characterized by a high mitotic activity, Fr1 DS thymocytes showed a reduced spontaneous proliferative capacity . When the expression of T cell receptor alpha- and beta-subunits was studied, the percentages of cells stained with anti-alpha and anti-beta antisera were found to be reduced in DS unfractionated thymocytes . The reduced number of high-density CD1+ thymocytes associated with a reduced spontaneous proliferative activity of low-density CD1+ thymocytes suggests that in DS thymuses there is a deficient expansion of immature T cells, resulting in a reduction of the various thymocyte subpopulations, including the thymocyte pool which differentiates into functionally mature T cells expressing the alpha-beta T cell receptor.

Cytometry, 1990, 11(8), 883 - 7
Simultaneous cytofluorometric analysis for the expression of cytoplasmic antigens and DNA content in CD3- human thymocytes; Zocchi MR et al.; We describe a method of two-color immunofluorescence staining which allows the simultaneous analysis of both cytoplasmic antigens and cell entry into the S/G2/M cell cycle phases . This analysis was performed on CD3(-)-activated thymocytes obtained from either highly purified CD1-CD3-CD4-CD8- cells or fresh thymus cell suspensions, stimulated with low doses of phorbol-12 myristate-13 acetate (0.5 ng/ml) and interleukin-2 . On the 14th day under these culture conditions about 90% of thymocytes did not express CD3 antigen on the cell surface . CD3- cells were further purified by cell sorting, fixed in paraformaldehyde, and permeabilized with Nonidet-P40 . Then these thymocytes were stained by indirect immunofluorescence with monoclonal antibodies identifying T cell-specific molecules (CD3, CD2, CD28, TCR alpha/beta, and TCR gamma/delta) and analyzed for DNA content . Interestingly, both CD3 and CD28 antigens were detectable in the cytoplasm of most cells (greater than 80%) . Further, the majority of the thymocytes which had entered the S/G2/M phases of the cell cycle (20%) expressed intracellular CD3 and CD28 molecules and reacted with the anti-beta framework beta F1 monoclonal antibody . The relationship between the appearance of CD3 and other T cell markers in the cytoplasm, the cell cycle entry, and the thymocyte development is discussed.

Cell Immunol, 1990 Jan, 125(1), 254 - 60
Tissue distribution and appearance in ontogeny of alpha/beta T cell receptor (TCR2) in chicken; Vainio O et al.; We have performed immunoperoxidase staining on cryostat tissue sections and immunofluorescence analysis on cell suspensions to identify cells expressing the alpha/beta T cell antigen receptor during ontogeny and adult life in chickens . We used the mouse monoclonal antibody, TCR2, which was previously shown to recognize the alpha/beta TCR in chickens . TCR2+ cells were observed in thymic cortex and medulla and in T-dependent areas of spleen, intestine, and cecal tonsils of young adult chickens . Some TCR2+ cells were found in the cortex of bursal follicles and in liver . The first TCR2+ cells appear in thymus on Day 13 of the embryonic life and it is only after hatching that TCR2+ cells begin to migrate to the periphery.

Dev Immunol, 1990, 1(1), 59 - 66
Are the IL-2 receptors expressed in the murine fetal thymus functional?
Zuniga-Pflucker JC, Smith KA, Tentori L, Pardoll DM, Longo DL, Kruisbeek AM.
It is well established that the majority of murine fetal thymocytes (day 15 of gestation) express receptors for interleukin 2 (IL-2), but the functional significance of these IL-2 receptors (IL-2Rs) is not clear . In situ hybridization data show a developmentally regulated expression of IL-2 and IL-2R mRNA . IL-2 binding studies were performed on fetal thymocytes and the results show the presence of both high (kD approximately equal to 20 pM) and low (kD approximately equal to 10 nM) affinity IL-2Rs . These IL-2Rs are indeed functional: intact fetal thymic lobes (but not cell suspensions) cultured in IL-2 exhibited an in vitro proliferative response at 20 pM of IL-2, corresponding with the presence of a functional high-affinity IL-2R on fetal thymocytes . The IL-2-dependent growth was primarily observed in the IL-2R+ thymic subset, which contains the CD3-/CD4-/CD8- precursor thymocytes . Furthermore, in vitro blocking of IL-2 in intact fetal thymic lobes resulted in a reduction in the cell yield, which predominantly affected the expansion of the immature CD3-/CD4-/CD8- thymocytes . Our findings strongly support the concept that the IL-2/IL-2R pathway is responsible for the proliferation of precursor cells within the fetal thymus.

Neuroscience, 1990, 37(2), 301 - 15
Fetal striatal neurons grafted into the ibotenate lesioned adult striatum: efferent projections and synaptic contacts in the host globus pallidus; Wictorin K et al.; The efferent projections to the host brain from intrastriatal grafts have been examined at the ultrastructural level . Cell suspensions of E14 rat fetal striatal tissue were implanted into the ibotenic acid lesioned caudate-putamen of adult rats . After survival times of at least five months, the anterograde neuronal tracer Phaseolus vulgaris leucoagglutinin was injected into the grafts . Consistent with our previous light microscopical analysis, anterogradely labelled fibres could be followed from the grafts into the host globus pallidus along the normal trajectory of the striatopallidal pathway in the internal capsule, and a few fibres also reached the entopeduncular nucleus and the substantia nigra . In the electron microscope, the graft-derived efferents were seen to be myelinated as they projected caudally along the fibre bundles of the internal capsule, and they were thus similar to the neostriatal projection in normal control animals . In the host globus pallidus, the graft-derived fibres ramified into a terminal network forming morphologically normal synaptic contacts with neuronal elements in the host globus pallidus . A total of 118 synaptic contacts were identified, all of them formed symmetric membrane specializations . The major postsynaptic targets were dendritic shafts (90%), with only a few contacts with spines or small shafts (8%) and perikarya (2%) . The morphology of the synaptic contacts and their distribution on different postsynaptic targets were similar to that which was found in the globus pallidus after tracer-injections into the caudate-putamen of normal control animals . The results show that grafted fetal striatal neurons can grow along the myelinated territory of the internal capsule to reinstate a seemingly normal synaptic input to the previously denervated neurons in the host globus pallidus.

Braz J Med Biol Res, 1990, 23(6-7), 567 - 71
Depressed adjuvant arthritis in rats transferred with spleen cells from Trypanosoma cruzi-infected syngeneic donors; Revelli SS et al.; In the present study we investigated whether the attenuating effect of chronic Trypanosoma cruzi (Tc) infection on adjuvant arthritis (AA) in the rat could be transferred to naive recipients . Transfer of whole spleen cells, but not of serum, from Tc-infected rats reduced AA (means +/- SEM: 11 +/- 0.5) in recipient animals (control values, means +/- SEM: 19 +/- 0.7) . Transfer of a T-cell-enriched subpopulation from spleen cells of Tc-infected rats (obtained by filtration through a nylon wool column) resulted in a similar attenuation of AA (means +/- SEM: 7.5 +/- 2.2) . The arthritic response of rats intraperitoneally inoculated with 2 x 10(5) Tc 48 h before induction did not differ from that observed in controls . Neither parasites nor specific antibodies were observed in suckling mice inoculated with serum or cell suspensions employed in transfer experiments . Consequently, the depressive effect on AA could not be directly attributed to Tc per se . We hypothesize that a homeostatic immunosuppressor mechanism may be responsible for this phenomenon.

Folia Biol (Praha), 1990, 36(2), 102 - 7
Ontogeny of MHC class II antigens in pigs; Trebichavsky I et al.; Cells constitutively expressing MHC class II antigens have been studied in the course of prenatal development in the Minnesota miniature pig . Frozen sections, cell suspensions and peripheral blood mononuclear cells were examined by using the HL-40 monoclonal antibody cross-reactive with a light chain determinant of the SLA-D molecule (MHC class II porcine antigen) . It could be demonstrated that the yolk sac contained cells expressing SLA-D antigens as early as the 24th day, the liver and the spleen on the 39th day of gestation . Splenic cells bearing SLA-D molecules formed periarteriolar structures . Except the spleen, peripheral blood, lymph nodes, bone marrow and intestinal wall were the main sites of SLA-D expression in the perinatal period of the pig.

J Endocrinol Invest, 1990 Jan, 13(1), 13 - 8
TRH raises cytosolic Ca2+ in human adenomatous lactotrophs; Spada A et al.; The effect of TRH on cytosolic free calcium concentrations, {Ca2+}i, was evaluated on cell suspensions obtained from 6 human PRL secreting pituitary adenomas . In these cells resting {Ca2+}i levels were variable (mean +/- SE; 103.8 +/- 6.5, n = 25); the addition of 100 nM TRH caused a marked {Ca2+}i rise within 20 sec., the peak values ranging from 200 to 437 nM (285 +/- 10.8 nM, n = 10) . The transients induced by TRH were composed by a rapid increase, due to mobilization of calcium from intracellular stores, followed within a few seconds by a lower plateau which was due to stimulated influx from the extracellular space . In fact, when EGTA and verapamil were applied after TRH they caused the Ca2+ plateau to dissipate rapidly . The addition of 1 microM dopamine (DA) caused a substantial decrease of resting {Ca2+}i (about 10-30%) as well as an inhibition of the plateau phase induced by TRH . The effect of DA completely depended on extracellular Ca2+ . The TRH-induced transients observed in adenomatous cells were quite similar in size and time course to those recorded in normal rat lactotrophs . As previously observed in rat lactotrophs, in adenomatous cells treatment with pertussis toxin (PTx, 1 microgram/ml for 4 h) was unable to affect the {Ca2+}i transients induced by TRH while completely abolished the effect of DA . The effects of TRH on in vivo and in vitro PRL secretion were also evaluated . Before surgery, no patient showed a positive response to the iv administration of 200 micrograms TRH (serum PRL levels: 95 +/- 62 ng/ml in basal conditions vs 124 +/- 92 after TRH, P = NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Mod Pathol, 1990 Jan, 3(1), 54 - 60
Comparison of histologic nodal reactive patterns, cell suspension immunophenotypic data, and HIV status; Westermann CD et al.; While cell suspension immunophenotypic studies are widely used as an aid in the diagnosis and classification of lymphomas and leukemias, much less attention has been directed toward interpretation of the results in reactive lymphoid proliferations . Cell suspension immunophenotypic data were therefore analyzed for 119 lymph nodes with reactive lymphoid proliferations which were divided into five major histologic categories: follicular hyperplasia, marked (FH,M), or moderate (FH); dermatopathic lymphadenopathy (DL); diffuse hyperplasia (DH); or "other." With the aid of a computer-assisted morphometer, the following were also measured and calculated: proportion of node occupied by follicles, mean relative follicle size, and mean follicle shape factor . Finally, in 57 cases, the influence of human immunodeficiency (HIV) status on the findings was analyzed . Although individual cases varied widely, cases of DL had significantly more CD3+ (T) cells, higher CD4:CD8 ratios, and fewer CD19+ (B) cells than other categories . Cases of FH,M had significantly lower CD4:CD8 ratios and more CD19+, CD10+, and transferrin receptor positive cells . Cases of FH,M and FH known to be HIV-negative had higher CD4:CD8 ratios than the HIV-positive cases . Peripheral blood CD4:CD8 ratios performed in 38 patients showed a strong correlation with nodal ratios . Morphometric data supported the correlation between follicular hyperplasia and increased proportions of CD19+, CD10+, and transferrin receptor-positive cells . Rare cases had CD5:CD2 or CD3 ratios of greater than 1 or "monoclonal" kappa to lambda ratios . CD4:CD8 ratios varied widely, but aberrant T cell phenotypes were not identified . These studies demonstrate that, although great variation exists, there are certain associations between types of reactive lymphoid hyperplasia and cell suspension immunophenotypic findings.(ABSTRACT TRUNCATED AT 250 WORDS)

Chin J Biotechnol, 1990, 6(2), 119 - 23
Plant regeneration from protoplasts of a super Chinese rice (Oryza sativa L.) cultivar--Zhonghua NO.8; Li JX et al.; Green rice plants were regenerated from protoplasts, which were derived from cell suspension of Oryza sativa L . cut . Zhonghua No . 8, a super Chinese rice cultivar with high productivity, good quality and high resistance to both bacterial blight and blast . The embryogenic calli were initiated from mature embroys . It took about 4 months to establish the cell suspenion . The regeneration plants from protoplasts were obtained in 2 months after the isolation of protoplasts.

Int Arch Allergy Appl Immunol, 1990, 93(2-3), 205 - 11
Increased expression of high-affinity low-density lipoprotein receptors on human T-blasts; Huber LA et al.; Like all cells, lymphocytes need cholesterol for proper function, a requirement met by a finely tuned homeostasis between intracellular synthesis and uptake from the environment via low-density lipoproteins (LDL) . We used flow cytometry to analyze the receptor activity of resting cells and T blasts incubated/activated in serum-free culture medium, or in medium supplemented with 25-5,000 micrograms/ml LDL . Dioctadecyl-indocarbocyanine has proved to be a useful fluorescent probe for investigating the LDL receptor activity of lymphocytes . The results show the receptor activity of day-3 resting T cells to be reduced more than 50% by 50 microgram LDL/ml, whereas 100-fold higher concentrations are necessary to achieve the same level of reduction in day-3 PHA blasts . The LDL receptor activities of individual blood donors' resting T cells, in vitro cholesterol-deprived resting T cells, and activated T blasts, were compared using two analytical techniques: spectrofluorometric analysis of detergent-solubilized cell suspensions and flow cytometric analysis of single living cells . Receptor affinity was determined by Scatchard analysis of spectrofluorometric binding curves, and by Line-weaver-Burke plots of flow cytometric data . Both methods yielded essentially identical dissociation constants (Kd) for cholesterol-deprived resting T cells and mitogen-activated T blasts, which fell in the expected range for the high-affinity LDL receptor (4.1-8.9 nM) . In addition, spectrofluorometric analysis, but not flow cytometry, permitted quantification of LDL uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Biomed Biochim Acta, 1990, 49(12), 1157 - 64
Liposome mediated in vitro transfection of pancreatic islet cells; Welsh N et al.; The aim of this study was to evaluate the suitability of the liposome technique for transfection of pancreatic islet cells in vitro . For this purpose, fetal islets were isolated and cultured free floating for two days after which they were further cultured, either intact or dispersed into islet cell suspensions, with different DNA-liposome preparations . The DNA-construct used were the control plasmid (pSP65) and the viral oncogene v-src contained in the plasmid pSPRIsrc . A previous study showed that islet cells transfected by means of electroporation with pSPRIsrc displayed an increased thymidine incorporation rate, making this plasmid suitable for further transfection studies . The DNA was associated with the liposomes by means of surface adhesion . The liposomes used were either conventional phosphatidylcholine-containing liposomes, phosphatidylethanolamine/oleic acid containing liposomes (pH-sensitive liposomes) or Lipofectin . After the exposure of islet cells to the DNA-liposome preparations, the transfection efficiency was assessed by determination of the uptake of the DNA-constructs (Southern blot analysis) and expression of the gene construct into an mRNA (Northern blot analysis) . In addition, the impact of the different DNA-liposome preparations on islet DNA replication (thymidine incorporation rates) was determined . It was found that two days after exposure to the DNA-liposomes, the v-src construct was located in islet cell nuclei and that v-src derived transcripts were transiently expressed in the islet cells . The Lipofectin liposomes were more efficient in transfecting islet cells than the pH-sensitive liposomes as assessed by the blotting techniques.(ABSTRACT TRUNCATED AT 250 WORDS)

Acta Biol Hung, 1990, 41(1-3), 257 - 65
Continuous induction of unscheduled DNA synthesis by gamma irradiation; Weniger P et al.; The induction of DNA-synthesis in non-S-phase cells is a very sensitive measure of a preceding damage of DNA . Usually, in an in vivo-in vitro test (treatment of an animal, incorporation of H3-thymidine in a cell suspension) the damaging of DNA takes place hours to days before the evaluation . In this case, the time course of the UDS-induction after a single dose of 1 Gy gamma irradiation was observed over a long period of time (21 months) . C57 black mice served as test animals . In an age of about 80 days they were irradiated and the induction of unscheduled DNA synthesis was measured at ten time intervals during the whole life-span of the animals . Although the repair in this gamma radiation damage in DNA is a very quick process--with centrifugation in alkaline sucrose a half-life of some minutes is found--an induction of unscheduled DNA synthesis could be seen at the irradiated animals until the end of their life (640 days) . The reason for this could be permanent disorders in cellular regulation caused by the gamma irradiation.

Horm Metab Res Suppl, 1990, 25, 82 - 7
A fluorometric viability assay for single human and rat islets; London NJ et al.; A microfluorometric viability assay for isolated human and rat islets of Langerhans has been developed using the fluorochromes fluorescein diacetate and propidium iodide . Fluorescein diacetate causes live cells to fluoresce green under blue light excitation (490 nm); propidium iodide causes dead cells to fluoresce red under green light excitation (454 nm) . The fluorescence intensity from the live and dead cells within a single islet was selectively measured by photometry using 520 nm and 610 nm barrier filters with blue and green light excitation respectively . All measurements were corrected for background fluorescence . The proportion of dead cells within single human or rat islets measured by microfluorometry was found to correlate highly significantly (r = 0.99, P less than 0.001) with the proportion of dead cells measured by dissociating the same islet into a single cell suspension and counting the actual proportion of dead cells . This assay therefore provides a rapid, accurate and objective measurement of the proportion of dead cells within isolated human and rat islets.

Stereotact Funct Neurosurg, 1990, 54-55, 328 - 36
Grafting of embryonic motoneurons into spinal cord and striatum of adult mice; Demierre B et al.; The aim of the present work was to determine whether embryonic motoneurons could survive in the adult CNS and whether they display different survival and growth characteristics in their natural site (the spinal cord) as compared to an ectopic site (the striatal region of the brain) . Specific labelling of embryonic motoneurons was obtained by retrograde transport of a fluorescent tracer (carbocyanine) followed by partial purification of the dissociated motoneurons on a density gradient . This technique offers the advantage that only the motoneurons in the cell suspension used for the grafts contain the fluorescent tracer . This study demonstrates that these motoneurons can survive and differentiate in the white and grey matters, not only in the adult spinal cord, but also in the brain . Furthermore, these motoneurons can migrate approximately 2 mm in the spinal cord and 4 mm in the brain.

Haematologia (Budap), 1990, 23(3), 151 - 9
Fibronectin and the adhesive properties of rat lymphocytes obtained from different peripheral lymphoid tissues; Altankov G et al.; A comparative investigation has been carried out on the effect of plasma fibronectin (Fn) on the adhesive properties of normal rat lymphocytes obtained from different lymphoid tissues: blood, spleen, mesenteric and tonsillar lymph nodes . Fn was immobilized on the basis of its ability to bind to gelatin . We established that concentrations of 40-50 micrograms/ml are sufficient for a saturation effect on Fn coating . For spleen cells an adhesion of 55.7 +/- 9.3%, for mesenteric lymph nodes 34.5 +/- 8.7% and for tonsillar cells 33.8 +/- 3.2% was observed . Blood lymphocytes showed the lowest adhesion, 21.3 +/- 4.2% . Compared to the other lymphoid tissues, the spleen cells exhibited a "basal" adherence to surfaces coated with gelatin only: 19.2 +/- 4.1% . T lymphocytes participate to a greater extent in the process, since their number was significantly reduced in cell suspensions after adhesion to both gelatin and gelatin-Fn coated surfaces . The addition of soluble Fn leads to a competitive inhibition of the lymphocyte adhesion to gelatin-Fn coated surfaces . The data demonstrated the important role of Fn for the adhesive interactions of lymphocytes during their functional distribution in the tissues.

Pathobiology, 1990, 58(5), 241 - 8
Reactivity of human tissues with monoclonal antibodies to myeloid activation and differentiation antigens . An immunohistochemical study; Koch AE et al.; We have characterized antimyeloid monoclonal antibodies (mAbs) produced to human rheumatoid arthritis (RA) synovial tissue macrophages (MPs) (8D7) and to lipopolysaccharide (LPS)-treated U937 cells (3D8) . The 3D8 antigen is upregulated with LPS stimulation of monocytes/MPs and during monocyte maturation . The 8D7 antigen is upregulated on functionally distinct subpopulations of RA synovial tissue MPs . We used immunohistochemistry to determine the spectrum of reactivity of these unique mAbs on myeloid cell suspensions, monocytes, and mature tissue inflammatory and noninflammatory MPs . The antigens identified by the mAbs were characterized biochemically, by immunoprecipitation of solubilized 125I-labelled antigens from cell surfaces, and immunohistochemically by enzymatic digestion of myeloid cells followed by a cellular ELISA . MAb 3D8, characterized as an anti-CD13 antibody, recognizes a 150-170 kd antigen, has almost exclusive myeloid reactivity, but reacts with Langerhans' cells of the skin and thymus, pointing to shared antigens between these cells and MPs . Unlike 3D8 antigen, 8D7 antigen is strongly expressed in inflammatory states, being present on MPs in granulomata as well as in sarcoid lymph nodes . Both mAbs react with frozen and methanol-Carnoy's fixed, paraffin-embedded tissues and detect antigenic differences among human mononuclear phagocytes present in different anatomical sites and in varying stages of differentiation and activation . These mAbs should prove to be a valuable tool for studying heterogenous populations of myeloid cells.

Virchows Arch B Cell Pathol Incl Mol Pathol, 1990, 59(5), 281 - 9
Collagenase of hepatocytes and sinusoidal liver cells in the reversibility of experimental cirrhosis of the liver; Montfort I et al.; In order to explore the cellular source(s) and the behaviour of the collagenolytic activity previously described in rat liver homogenates, in the reversibility of experimental cirrhosis of the liver, enriched suspensions of hepatocytes and of sinusoidal liver cells were obtained by a procedure which employs low EDTA concentrations and no bacterial collagenase . Cell suspensions were prepared from three different groups of animals: 1) normal controls, 2) rats with CCl4-induced cirrhosis of the liver, and 3) rats with swine serum-induced cirrhosis of the liver . Animals were sacrificed in each group upon completion of treatment and also after 3, 6 and 12 months . In each liver wet weight and collagen concentration were determined, and collagenolytic activity of both enriched cell suspensions was measured separately . In addition, histological studies of liver tissue and ultrastructural examination of cell suspensions were performed by standard procedures . Enriched suspensions of both normal hepatocytes and sinusoidal liver cells display Ca2(+)-dependent collagenolytic activities . Both cell suspensions obtained from each of the two types of cirrhotic livers show normal or slightly increased average levels of collagenase activity at the time of treatment discontinuation, when average liver collagen content ranges from 6 to 10-fold over normal, suggesting that the normal collagenase/collagen ratio is disturbed and that collagenolytic activity is deeply decreased in relation to the actual liver collagen load.(ABSTRACT TRUNCATED AT 250 WORDS)

Virchows Arch B Cell Pathol Incl Mol Pathol, 1990, 59(5), 263 - 70
Lymphocytes from hepatic inflammatory infiltrate kill rat hepatocytes in primary culture . Comparison with peripheral blood lymphocytes; Ramadori G et al.; In the last few years it has become possible in the liver to isolate lymphocytes from inflammatory infiltrates and to culture them in vitro . Most of the lymphocyte clones obtained are CD 8+ cytotoxic cells, but interactions between these lymphocytes and hepatocytes in primary culture have not been analysed previously . In this study, cloned human T lymphocytes from liver biopsies and from the peripheral blood of patients with chronic hepatitis B or primary biliary cirrhosis, after phenotypical and functional characterization into CD 8+ or CD 4+ cytotoxic lymphocytes, were activated in an antigen-independent fashion by adding either anti CD 3 or anti CD 2/R-3 monoclonal antibodies to the cell suspension . The activated cells were then coincubated with rat hepatocytes in primary culture . The killing capacity of the activated lymphocytes was monitored by light and electron microscopy and by measurement of lactic dehydrogenase (LDH)-release into the culture medium . It was found that cytotoxic CD 8+, but not CD 4+ helper lymphocytes very effectively killed hepatocytes . The killing effect was dependent on the time of cocultivation and on effector-target (E/T) ratio . Total breakdown of the hepatocyte monolayer was achieved after 10-20 h coculture and at an E/T ratio of 10 to 1 . As LDH-release in the culture medium reached about 80% of the total LDH-content, most of the hepatocytes were lysed by activated lymphocytes . Cytotoxic activity of clones obtained from different biopsies was comparable with that of clones from peripheral blood . Hepatocytes in primary culture seem to be very sensitive to the killing capacity of activated cytotoxic lymphocytes.

Exp Brain Res, 1990, 81(2), 433 - 7
Seizure suppression in kindling epilepsy by intrahippocampal locus coeruleus grafts: evidence for an alpha-2-adrenoreceptor mediated mechanism; Bengzon J et al.; Intrahippocampal cell suspension grafts, prepared from the locus coeruleus region of rat fetuses, have previously been shown to retard seizure development in rats made hypersensitive to hippocampal kindling by a lesion of the forebrain noradrenergic system . The objective of the present study was to provide evidence that the seizure-suppressant effect elicited by the grafts is mediated via noradrenergic mechanisms . Two groups of rats received 6-hydroxydopamine in the lateral ventricle and then bilateral intrahippocampal locus coeruleus grafts . After 3 months, the grafted animals and a group of normal rats were subjected to hippocampal kindling . One group of grafted animals and the normal rats were injected intraperitoneally with the alpha-2 adrenergic receptor blocker idazoxan before each kindling stimulation . The other grafted rats received vehicle injections . The development of seizures was significantly faster in the grafted and normal rats that had been given idazoxan than in the grafted rats that had not been subjected to alpha-2 receptor blockade . Our data suggest that the seizure-suppressant action exerted by grafts of fetal locus coeruleus in hippocampal kindling is mediated via noradrenergic mechanisms, most likely via activation of postsynaptic alpha-2 adrenoreceptors.

Vrach Delo, 1990 Jan, (1), 90 - 3
{Cryopreservation of a suspension of fetal liver cells for clinical use}; Lavrik SS et al.; The authors evaluate a method of procuring and preserving fetal liver cells in a medium containing low-molecular polyvynilpyrrolidon, glucose, lactose, sodium phosphate and sodiumhydrocitrate not requiring washing off . The biomaterial is stored at a temperature of -196 degrees C . The efficiency of this method of cryopreservation of fetal liver cell suspensions was confirmed by high morphological intactness, functional activity and proliferative capacity . Patients with hypoplastic anemia subjected to transfusion of cryopreserved fetal liver cells showed a normalization of bone marrow hemopoiesis, remissions.

Exp Brain Res, 1990, 79(1), 3 - 17
Intrastriatal implants of mesencephalic cell suspensions in weaver mutant mice: ultrastructural relationships of dopaminergic dendrites and axons issued from the graft; Triarhou LC et al.; Dissociated cell suspensions were prepared from the ventral midbrain of normal mouse foetuses and stereotaxically implanted into the neostriatum of 2-3 months old homozygous weaver mutant mice, which are severely deficient in dopamine . In tests of amphetamine-induced turning behaviour 60 days after grafting, recipient animals displayed a rotational bias opposite to the grafted side . Prior to perfusion, which was carried out at 80 days after transplantation surgery, the grafted striata of the weaver recipients were deprived of their intrinsic mesostriatal dopamine input by local injections of 6-hydroxydopamine into the ipsilateral substantia nigra in order to selectively study the innervation derived from the graft . Grafts were found to contain an estimated 100-700 tyrosine hydroxylase immunoreactive neurones . An ultrastructural analysis demonstrated that both axons and dendrites immunoreactive for tyrosine hydroxylase extended from the graft into the recipient striatum . In the host striatum proximal to the graft (i.e . at a distance of 0.0-0.5 mm from the graft) the proportion of dendrites to axons was about 1:2, whereas distal to the graft (i.e . at a distance of 0.5-1.0 mm) it was 1:20 . Graft-derived tyrosine hydroxylase immunoreactive axons were primarily found in apposition with unlabelled dendrites or spines of the recipient striatum (greater than 90%) . Graft-derived dopaminergic dendrites received synaptic input from unlabelled axon terminals and were opposed to the unlabelled somata of striatal neurones in a few instances . In conclusion, this study shows that mesencephalic cell suspensions survive in the weaver striatum and provide a functional dopamine innervation which comprises both axonal and dendritic processes.

Cancer Immunol Immunother, 1990, 31(1), 19 - 27
Expression of the adhesion molecule ICAM-1 and major histocompatibility complex class I antigens on human tumor cells is required for their interaction with autologous lymphocytes in vitro; Vanky F et al.; In a group of 30 human tumors, comprising 12 lung, 14 ovarian, 2 breast carcinomas, 1 hypernephroma and 1 mid-gut carcinoid, the expression of major histocompatibility complex (MHC) class I molecules and the intercellular adhesion molecule 1 (CAM-1, CD54) was found to vary independently . Some tumors expressed both or neither of these molecules . Among 9/13 ICAM-1+ tumors, in which greater than 50% cells reacted with the anti-ICAM-1 monoclonal antibody (mAb) (LB-2), the class I antigen was also detected on greater than 50% of the cells . Only 2 ICAM-1+ tumors were class-I- . In 5/17 cases the tumors were MHC-class-I+ and ICAM-1- . Lymphocytes collected from the blood or from the tumor site were assayed for recognition on the tumor cells in the auto-tumor cytotoxicity test and in mixed lymphocyte tumor cell culture (MLTC) . Positive results were obtained only with the MHC-class-I+/ICAM-1+ tumors . In vitro treatment of the tumor cell suspensions with interferon gamma and tumor necrosis factor alpha (TNF alpha) induced or enhanced the ICAM-1 and/or class I antigen expression in 8/12 cases . Of the tumor samples treatged, 8/9 acquired stimulatory capacity and 3/10 became susceptible to lysis by the lymphocytes . In 6/6 MLTC performed with the cytokine-treated tumor cells, cytotoxicity against the autologous tumor was generated . Three of these MLTC lymphocytes also lysed the untreated targets . mAb directed to class I antigens or to ICAM-1 inhibited both the stimulation by and the lysis of tumor cells when confronted with fresh lymphocytes . The cytotoxicity generated in the MLTC was also inhibited . If, however, the cytotoxic function was induced in MTLC containing interleukin-2 (5 U/ml), inhibition was obtained only by pretreatment of the targets with mAb against ICAM-1 . The results show thus (a) that the lymphocytes react in vitro with tumor cells only if these express both MHC class I molecules and ICAM-1; (b) that expression of these molecules can be induced by interferon alpha and TNF alpha; (c) that cytotoxic effectors generated in the MLTC with cytokine-treated tumors can also act on the untreated tumor cells . The requirement of the two surface moieties for the interaction with lymphocytes was also substantiated by blockade with relevant mAb.

Biochem J, 1990 Jan 1, 265(1), 39 - 44
Identification and characterization of somatostatin receptors in neonatal rat long bones; Bruns C et al.; Somatostatin (somatotropin release inhibiting factor; SRIF) has widespread functions as a modulator of neural activity as well as of endocrine and exocrine secretion . In the present paper, the binding characteristics of somatostatin receptors have been investigated in rat long bones using the stable analogue, 125I-SDZ 204-090, as a ligand . Binding studies revealed the presence of a single class of high-affinity binding sites for 125I-SDZ 204-090 on cells prepared from neonatal rat long bones with an equilibrium dissociation constant (KD) of 70.1 +/- 8.2 pM (n = 3) . An excellent correlation was found between the ability of various somatostatin analogues to inhibit growth hormone in pituitary cells and to displace the binding of 125I-SDZ 204-090 to the bone cell preparation, indicating that the receptors are very similar, if not identical . The localization of the somatostatin-binding sites was examined by autoradiography after labelling in vitro and in vivo . The binding sites were shown by both procedures to be selectively localized to the metaphysis of rat long bones . The labelling experiments in vivo indicate that these receptors can be reached in the living animal by circulating somatostatin analogues . In addition, the analogue SMS 201-995 inhibited the forskolin-stimulated adenylate cyclase activity in bone cell suspensions . These results suggest that somatostatin could be an important regulatory factor in bone metabolism.

Int J Radiat Biol, 1990 Jan, 57(1), 103 - 15
Bone marrow from Balb/c mice radiocontaminated with 241Am in utero shows a deficient in vitro haemopoiesis; Van Den Heuvel RL; Radiation damage from 241Am to bone marrow cells was manifest in long-term bone marrow cultures (LTC) from offspring of mice radiocontaminated at the 14th day of gestation (119, 479, 803, 1754 kBq 241Am/kg) . Offspring were reared by their own contaminated mother for 3 weeks postnatal . LTC from these offspring were less able to support in vitro CFC proliferation than control LTC from non-contaminated offspring . This radiation damage persisted 71 weeks after radiocontamination in utero . Using this in vitro culture system, damage was observed at lower doses if 241Am contamination occurred at foetal than at adult ages . Radiation damage was observed only using LTC, while the haemopoietic stem cell concentration (CFU-S, in vitro CFC) and the stromal stem cell concentration (CFU-F) from marrow in situ were not impaired after 241Am radiocontamination in utero . After culturing LTC in 25 per cent FCS and recharging the stromal adherent layer with bone marrow cell suspensions originating either from control offspring or from offspring contaminated with 241Am in utero, some evidence was found that the proliferation capacity of the haemopoietic cells was diminished . However, the nature of effects on the stromal elements is currently somewhat equivocal . Following in utero contamination the stromal adherent cells appeared to support better the production of in vitro CFC.

J Immunol, 1990 Jan 1, 144(1), 7 - 15
Antibodies against the CD44 p80, lymphocyte homing receptor molecule augment human peripheral blood T cell activation; Denning SM et al.; The CD44 inhibitor Lutheran {In(Lu)}-related p80 molecule has recently been shown to be identical to the Hermes-1 lymphocyte homing receptor and to the human Pgp-1 molecule . We have determined the effect of addition of CD44 antibodies to in vitro activation assays of PBMC . CD44 antibodies did not induce PBMC proliferation alone, but markedly enhanced PBMC proliferation induced by a mitogenic CD2 antibody pair or by CD3 antibody . CD44 antibody addition had no effect upon PBMC activation induced by PHA or tetanus toxoid . CD44 antibody enhancement of CD2 antibody-induced T cell activation was specific for mature T cells as thymocytes could not be activated in the presence of combinations of CD2 and CD44 antibodies . CD44 antibody enhancement of CD2-mediated T cell triggering occurred if CD44 antibody was placed either on monocytes or on T cells . In experiments with purified monocyte and T cell suspensions, CD44 antibodies A3D8 and A1G3 augmented CD2-mediated T cell activation by three mechanisms . First, CD44 antibody binding to monocytes induced monocyte IL-1 release, second, CD44 antibodies enhanced the adhesion of T cells and monocytes in CD2 antibody-stimulated cultures, and third, CD44 antibodies augmented T cell IL-2 production in response to CD2 antibodies . Thus, ligand binding to CD44 molecules on T cells and monocytes may regulate numerous events on both cell types that are important for T cell activation . Given that recent data suggest that the CD44 molecule may bind to specific ligands on endothelial cells (vascular addressin) and within the extracellular matrix (collagen, fibronectin), these data raise the possibility that binding of T cells to endothelial cells or extracellular matrix proteins may induce or up-regulate T cell activation in inflammatory sites.

J Clin Lab Immunol, 1990 Jan, 31(1), 27 - 31
Inhibition of in vitro plaque formation by large granular lymphocyte leukemia cells from F344 rats; Stromberg PC et al.; The effect of large granular lymphocyte leukemia on B lymphocyte function was studied by determining the number of plaques formed in an in vitro hemolytic plaque assay . Leukemia cells inhibited plaque formation by normal splenic lymphocytes in a logarithmic, dose-dependent manner . At the highest leukemia cell concentrations, spleen cell suspensions made 50% fewer plaques . Plaque forming responses were very sensitive to duration of preincubation time in all assays . The number of plaques formed decreased markedly if incubated 2 hr before the assay was performed . Incubation of the cells at 56 degrees C for 8 min did not alter the inhibitory activity but pretreatment with 0.01% trypsin did . Supernatant fluids from leukemia cell suspensions did not inhibit plaque formation . These data suggest that diffuse infiltration of lymphoid tissues by leukemia cells may interfere with some normal lymphocyte functions . Although leukemia cells inhibited splenic B lymphocyte function, leukemic rats did not have hypogammaglobulinemia.

Symp Soc Exp Biol, 1990, 44, 193 - 205
Phosphoinositides and plant growth substance action; Hanke DE et al.; Synergism between 0.5 mM inositol and cytokinin in the stimulation of plant tissue culture growth suggests a role for inositol in the mediation of cytokinin action . Investigation of the effects of cytokinin on the pattern of labelling of lipids from radioactive precursors by cytokinin-responsive cells of a cytokinin-dependent soybean cell suspension culture did not detect any reproducible link between cytokinin action and lipid labelling after 10 min . Evidence for links between auxin action and phosphoinositide metabolism in other systems would benefit from confirmation of long term repeatability and more rigorous chemical characterization of the compounds involved . For plant growth substance action to be mediated by release of inositol trisphosphate from phosphatidylinositol bisphosphate, activation of phospholipase C, possibly requiring potentiation by a GTP-binding protein, would be expected . Reports of G-protein effects on plant phospholipase C activity are conflicting . There is evidence for G-protein stimulation of activity from the results of assays using endogenous substrates, although the products released have not been fully characterized . Other results, from assays using exogenous substrates, have shown no effect of GTP analogues on the enzymic breakdown of phosphatidylinositol bisphosphate . Using endogenously labelled membranes from soybean cells, we were unable to detect effects attributable to G-protein potentiation . None of a range of growth substances at physiologically active concentrations proved able to alter detectably the lack of response of polyphosphoinositidase activity to GTP-analogues . The activity was, however, strongly stimulated by Ca2+ at micromolar levels, a characteristic widely reported . In consequence, the possibility that transient increases in the labelling of inositol phosphate fractions may be a result of increases in cytosolic Ca2+ levels needs to be addressed . If there is a role for inositol in soybean cell activation by cytokinin, we have no evidence that it involves polyphosphoinositide cleavage . That there is a special role for inositol in the mitotic cycle of soybean cells and in addition to the maintenance of viability is shown by the results of experiments in which endogenous inositol synthesis was inhibited . Further research aims to identify the inositol-requiring steps and their relationship, if any, with the auxin-requiring and cytokinin-requiring steps in the mitotic cycle of cultured soybean cells.

Neuroscience, 1990, 37(2), 353 - 66
Dissimilar responses of adult thalamic monoaminergic and somatosensory afferent fibers to implantation of thalamic fetal cells; Nothias F et al.; It is generally accepted that transplanted fetal neurons can, after several weeks to months, establish connections with the host CNS . Host afferent systems seem, however, to show different types of responses to the presence of grafted fetal neurons . The present study is a preliminary step to identify mechanisms involved in the reactions of adult axons to transplanted fetal neurons . The right ventrobasal thalamus of adult rats was depleted of neurons by in-situ injection of kainic acid and cell suspensions from homotopic thalamic embryonic primordia which were injected into the lesioned area . After various post-implantation delays, ranging from five to 30 days, two types of experiments were performed: (i) noradrenaline and serotonin immunohistochemistry with specific antibodies on alternate sections; and (ii) anterograde tracing using wheat germ agglutinin conjugated to horseradish peroxidase from the dorsal column nuclei and the principal sensory trigeminal nucleus . Five days after transplantation, host monoaminergic fibers (either noradrenergic or serotoninergic) had already grown into the transplants . Ingrowing fibers were thin and poorly varicose, exhibiting endings morphologically similar to the growth cones observed during axogenesis . Seven days after grafting, growth cones were no longer visible and monoaminergic fibers exhibited either normal-sized or very large varicosities . Large varicosities progressively decreased in number and, after three weeks, the fibers displayed a normal adult morphology, forming a dense network all over the transplants . In contrast, host somatosensory afferents, labeled by anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase, did not grow into the transplants . Intermingling of somatosensory afferents and transplanted cells was observed only after 10 days, when grafted neurons extended outside the original transplantation site into the neuron-depleted area containing the somatosensory afferents . The present results demonstrate that adult monoaminergic and somatosensory afferents, when deprived of their usual target, do not react in a similar way to the addition of fetal neurons . It is proposed that adult monaminergic fibers have the ability to regain morphological (and probably functional) immature forms which were considered to be restricted to the period of axogenesis or to lesion-induced regeneration . In contrast, fetal transplants do not seem to induce, by themselves, a similar alteration of genetic expression in adult somatosensory neurons . It has been proposed that "diffuse" and "point-to-point" axonal systems may be differentiated in the CNS on anatomical bases . The present results add to the identification of two different systems by demonstrating that, in the thalamus, they present dissimilar responses to the implantation of fetal cells.

Verh Dtsch Ges Pathol, 1990, 74, 432 - 6
{Induction of primitive neuroectodermal tumors by oncogene complementation}; von Deimling A et al.; Primitive neuroectodermal tumors (PNET) represent a family of undifferentiated neural neoplasms which predominantly occur in children . PNETs are likely to originate from precursor cells and exhibit a marked potential for neuronal, glial and ependymal differentiation . A prominent example is the medulloblastoma of the cerebellum . In a model system using a novel transgenic CNS transplantation model, we have introduced a combination of ras and myc oncogenes into cell suspensions from fetal forebrain (E14) and postnatal cerebellum (P2) of the rat . Oncogene transfer into fetal forebrain grafts resulted in a high incidence of anaplastic neural tumors predominantly derived from glial precursors . Cell lines established from these neoplasms expressed high levels of both oncogenes . In a second experiment, the ras/myc vector was introduced into cell suspensions from neonatal cerebellum . The transformed cells were cultured for 3 weeks . Following stereotaxic transplantation, tumors of a similar morphology were observed . However, one animal developed a neoplasm with features of a cerebellar medulloblastoma . A cell line which exhibits a marked capacity for neurite extension and synaptogenesis was established from this tumor . Since these cells neither express ras nor myc, an insertion mutagenesis event appears to be responsible . Experiments to characterize this mutation are in progress . Our results indicate a potent transforming effect of ras and myc on neural precursor cells in vivo.

Arkh Patol, 1990, 52(11), 62 - 3
{Method of bronchoalveolar lavage for performing cytochemical examination in chronic nonspecific pulmonary inflammation}; Grobova OM et al.; The method suggested includes the use of homogenizer terrilytin for removing excessive mucus from broncho-alveolar lavage (BAL) . Cytochemical investigation of BAL neutrophils is reasonable to be performed if the viability of the cell suspension is not lower than 60% . Cytospectrophotometric examination is recommended for an unbiased evaluation of the neutrophil functional state.

Stereotact Funct Neurosurg, 1990, 54-55, 364 - 7
Effects of interstitial edema on brain cell transplantation; Miyazaki S et al.; Fetal raphe cells were transplanted into the anterior part of the corpus callosum of serotonin denervated hydrocephalic rats using a cell suspension method . Hydrocephalus was induced by intracisternal injection of kaolin . The survival of the transplanted cells and fiber outgrowth were evaluated according to the level of serotonin and its metabolite, hydroxyindoleacetic acid, using high-performance liquid chromatography with electrochemical detection in the anterior and posterior parts of the corpus callosum 1-2, 5-6, and 7-8 weeks after transplantation . The results suggest favorable effects of interstitial edema associated with hydrocephalus on the survival of transplanted raphe cells and fiber outgrowth.

Lab Delo, 1990, (8), 31 - 2
{Determination of the erythrocyte count using a microcolorimeter}; Storozhuk PG et al.; Red cell count is estimated with the use of a MKMF-1 microcolorimeter . 10 ml of Gower's solution are used; 0.02 ml of blood are added to this solution . Photometry of red cell suspension is carried out in a cuvette with a 0.5 cm optic route at lambda 610 nm . Microampermeter data are read from the optic density D scale . The D value is substituted into the formula: red cell count (RC) = K.D.10(6), where K coefficient is found experimentally by measuring the D and by estimating red cell count in microscopy, and is equal to 33.3 . This formula permits estimating red cell count in 1 microliter of blood.

Lab Delo, 1990, (8), 27 - 9
{Determination of the rate of utilization of glucose in a suspension of erythrocytes}; Kunitsyn VG et al.; The authors suggest a new method for measuring the rate of glucose utilization in a cell (red cell) suspension, making use of interferometry . This method allows an easy examination of glucose utilization kinetics in time with but 1000 cells . Thermostatic control of the sample in the cuvette of the device permits detecting the temperature effects on glucose metabolism kinetics and thus define the temperature optimal for the process . A correlation was revealed between the point of structure phase transition in the red cell membrane and the optimal temperature for glucose utilization within the range of physiologic temperatures . This method may be useful in medical, biochemical, physiologic, and hematologic studies . The relative error of the technique is 3 percent.

Prog Brain Res, 1990, 82, 95 - 101
Angiogenesis and the blood-brain barrier in solid and dissociated cell grafts within the CNS; Broadwell RD et al.; Available evidence suggests that blood vessels indigenous to solid CNS and peripheral tissues grafted to the brain are sustained and maintain the morphological and permeability characteristics they manifest in normal life . Furthermore, these vessels of graft origin anastomose (albeit not rapidly) with vessels of the surrounding host tissue predominantly at the host-graft interface and less so, or not at all, within the graft itself . For these reasons, blood-brain and brain-blood barriers, evident in the late fetal and neonatal CNS, can be expected to exist within CNS grafts placed intracerebrally or extracerebrally, providing the graft remains viable . Peripheral neural and non-neural tissues not possessing cellular barriers to circulating macromolecules do not acquire such barriers subsequent to their transplantation within the CNS . The absence of a blood-brain barrier in the adrenal gland grafted intracerebrally may be relevant for the treatment of Parkinson's disease with blood-borne therapeutics . Compared to solid tissue grafts, cell suspension grafts have the potential of becoming vascularized rapidly . That cell suspensions of neurons and of glia are supplied with BBB vessels of host origin and that the permeability characteristics of host BBB vessels are altered by a tumor cell suspension reaffirm the belief that the type of transplanted cell/tissue indeed determines the permeability characteristics of the blood vessels supplying it . The suspected immunologic privilege of the CNS is not absolute . Eventual host rejection of allografts placed within the third ventricle may be a dual consequence of the absence of a BBB at the level of the host median eminence and involvement of the minor histocompatibility complex.

Lab Delo, 1990, (11), 17 - 9
{pH-metric study of ion equilibrium in erythrocyte suspensions exposed to heat}; Vinogradov AP; A method of differentiated measurements of pH is suggested to be used in study of the time course of ionic equilibrium in red cell suspensions exposed to heating . The described method yields more accurate and complete data on the type and distribution of red cells as regards their membrane characteristics.

Arch Dermatol Res, 1990, 282(2), 126 - 30
Flow cytometric quantification of human epidermal cells expressing keratin 16 in vivo after standardized trauma; de Mare S et al.; The intermediate filament protein keratin 16 is expressed in hyperproliferative epidermis . The present study aims to clarify the relationship between the expression of this keratin type, hyperproliferation (percentage of cells in SG2M phases), and keratinization (keratin 10 expression) . These three parameters were quantified in biopsy material taken at different time intervals following sellotape stripping--this being a dynamic in vivo model for the induction of hyperproliferation . From the biopsy specimens cell suspensions were prepared, labeled with antibodies KS8.12 (specially directed against keratin 16) and RKSE60 (directed against keratin 10), and analyzed using flow cytometry . Percentages of cells in SG2M phases were assessed by measuring the relative DNA content after propidium iodide staining . Keratin 16 expression in the suprabasal layer anticipated epidermal proliferation, suggesting a role of the suprabasal compartment in the induction of epidermal growth . Keratin 10 expression decreased about 1 day after the onset of keratin 16 expression, indicating that these processes do not depend directly upon each other.

Urol Res, 1990, 18(2), 131 - 6
Chemosensitivity testing of primary human renal cell carcinoma by a tetrazolium based microculture assay (MTT); Mickisch G et al.; MTT staining procedures have been used in chemosensitivity testing of established cell lines of human and other sources as well as of human leukaemias, but only limited information on its application in primary solid human tumors is presently available . We have evaluated MTT staining in primary human Renal Cell Carcinomas (RCCs), studied various factors interfering with the optimal use, and finally applied it in subsequent chemosensitivity testing . The method depends on the conversion of a water-soluble tetrazolium salt (MTT) to a purple colored formazan precipitate, a reaction effected by enzymes active only in living cells . Single cell suspensions of RCCs were obtained either by enzymatic dispersion or by mechanical dissagregation, filtered through gauze, and purified by Ficoll density centrifugation . Tests were carried out in 96-well microculture plates . 10(4) viable tumor cells per well at 4 h incubation time with 20 micrograms MTT/100 microliters total medium volume yielded best results . Formazan crystals were dissolved with DMSO, and the plates were immediately measured on a microculture plate reader at 540 nm . Under these criteria, linearity of the system could be demonstrated . For chemosensitivity testing, cells were continuously exposed to a number of drugs prior to the MTT staining procedure . Reproducibility of results was assessed and confirmed by culturing RCCs in flasks additionally, resubmitting them after 1, 2, and 4 weeks to the MTT assay . We conclude that the semiautomated MTT assay offers a valid, rapid, reliable and simple method to determine the degree of chemoresistance in primary human RCCs.

Biodegradation, 1990, 1(4), 253 - 61
Reductive dechlorination of 1,2-dichloroethane and chloroethane by cell suspensions of methanogenic bacteria; Holliger C et al.; Concentrated cell suspensions of methanogenic bacteria reductively dechlorinated 1,2-dichloroethane via two reaction-mechanisms: a dihalo-elimination yielding ethylene and two hydrogenolysis reactions yielding chloroethane and ethane, consecutively . The transformation of chloroethane to ethane was inhibited by 1,2-dichloroethane . Stimulation of methanogenesis caused an increase in the amount of dechlorination products formed, whereas the opposite was found when methane formation was inhibited . Cells of Methanosarcina barkeri grown on H2/CO2 converted 1,2-dichloroethane and chloroethane at higher rates than acetate or methanol grown cells.

Bioprocess Technol, 1990, 10, 251 - 70
Suspension culture of mammalian cells; Birch JR et al.; Mammalian cell suspension culture systems are being used increasingly in the biotechnology industry . This is due to their many advantages including simplicity and homogeneity of culture . Suspension systems are very adaptable (e.g., for microcarrier, microencapsulation, or other methods of culture) . Their engineering is thoroughly understood and standardized at large scale, and automation and cleaning procedures are well established . Suspension systems offer the possibility of quick implementation of production protocols due to their ability to be scaled easily once the basic culture parameters are understood . The only main disadvantage of the suspension culture systems to date is their inapplicability for the production of human vaccines from either primary cell lines or from normal human diploid cell lines (Hayflick et al., 1987 and references therein) . One of the great advantages of suspension culture is the opportunity it provides to study interactions of metabolic and production phenomena in chemostat or turbidostat steady-state systems . Furthermore, in suspension culture systems from which cell number and cell mass measurements are easy to obtain, rigorous and quantitative estimations of the effects of growth conditions or perturbations of metabolic homeostasis can be made . Such studies can speed up the development of optimal processes . With our increasing understanding of factors influencing expression in mammalian cells (Cohen and Levinson, 1988; Santoro et al., 1988) and the direct application of new methods in suspension culture (Rhodes and Birch, 1988), its usefulness and importance is likely to increase in the future . In this chapter, we have described some of the potential uses of the various suspension culture systems and have covered most of the established technology and literature . Due to the rapid developments and needs in the biotechnology industry and the versatility of suspension culture systems, it is probable that many more variations on this theme will evolve in the near future at both the pilot and production scales.

Phytochemistry, 1990, 29(7), 2149 - 52
Biotransformation of 18 beta-glycyrrhetinic acid by cell suspension cultures of Glycyrrhiza glabra; Hayashi H et al.; Two biotransformation products formed from 18 beta-glycyrrhetinic acid by cell suspension cultures of Glycyrrhiza glabra were isolated and their structures determined by chemical and spectral data as 3-O-{alhpa-L-arabinopyranosyl-(1----2)-beta-D-Glucuronopy ranosyl}-24- hydroxy-18 beta-glycyrrhetinic acid and 30-O-beta-D-glycopyrano-syl-18 beta-glycyrrhetinic acid . The formation of glycyrrhizin, the main triterpene glucuronide of the licorice root, was not detected among the biotransformation products . This is the first report of the glucuronylation of an exogenous triterpene in plant cell cultures.

Phytochemistry, 1990, 29(4), 1131 - 5
Induction of two prenyltransferases for the accumulation of coumarin phytoalexins in elicitor-treated Ammi majus cell suspension cultures; Hamerski D et al.; Two dimethylallyl diphosphate:umbelliferone dimethylallyltransferase (prenyltransferase) activities, catalysing the 6-prenylation and the 7-O-prenylation, respectively, of umbelliferone in the course of phytoalexin synthesis, increased in Ammi majus cell suspension cultures in response to elicitor treatment . Both enzyme activities were dependent on Mg2+ or Mn2+ with significant preference for Mg2+ in the 6-prenylation reaction . Whereas dark-grown cells did not contain these activities, both prenyltransferase activities were induced rapidly by the addition of elicitor reaching a first maximum after 10-14 hr and a second maximum beyond 30 hr . Other coumarin specific, elicitor-induced enzyme activities of A . majus cells, in contrast, showed only one maximum of activity within the 50 hr experimental period, while the pattern of induction of phenylalanine ammonia-lyase activity resembled that of the prenyltransferases with maxima at ca 8 hr and 20-30 hr . Preliminary data suggest that the apparent biphasic induction of these enzyme activities is due to post-translational enzyme modifications.

Tumori, 1989 Dec 31, 75(6), 542 - 6
Detection of the 170 kDa P-glycoprotein in neoplastic and normal tissues; Ronchi E et al.; A membrane purification procedure and an immunoblotting assay have been designed to allow screening of human solid tumors for overexpression of the GP170 glycoprotein without employing a disaggregation method to obtain cell suspensions . The electrophoresed membrane proteins were probed, after Western Blotting, with the C219 monoclonal antibody and iodinated Protein A . The labeling intensity of the bands on the autoradioimmunoblots were quantified by densitometry . To test for the presence of GP170, we used membranes from the UV 2237 fibrosarcoma line and its adriamycin-resistant variant ADMR, grown in vitro or as solid tumor in mice . Membranes of human normal and tumor tissues obtained from previously untreated patients were also tested . An immunoreaction was observed in the adriamycin-resistant UV 2237 lines grown in vitro or in vivo . Quantitatively, the binding of the resistant cell line grown in vitro was higher than that observed in cells grown in mice . Bands in the GP 170 region were observed in 4/7 normal and in 7/7 tumor colon tissues and in the normal medulla from 2 patients with cancer of the renal cortex . No reaction could be found in samples from normal tissue, primary tumor or nodal metastasis from 7 patients with breast cancer.

Tumori, 1989 Dec 31, 75(6), 570 - 5
In vivo and in vitro growth of SCLC cells derived from biopsies; Giani S et al.; In order to increase the availability of SCLC cells derived from biopsies, in vivo and in vitro growth methods were investigated . The cells grown in both conditions were periodically monitored for reactivity with 2 monoclonal antibodies (MAbs): MLuC1 directed against SCLC cells and IM1 which recognizes the class II antigen on activated lymphocytes and macrophages . About 50% of the 28 analyzed SCLC specimens were found to proliferate in one or both systems . The in vitro-grown cells exhibited the same heterogeneity found in the original cell suspensions and moreover, in some cases only normal cells were recovered after several in vitro passages . From the subcutaneous transplanted tumors a large number of MLuC1-positive tumor cells could easily be recovered, thus indicating the validity of the in vivo methodology . The MBr1 MAb, directed against an epithelial antigen, was found to react with about 50% of the 26 tested tumors, mainly those which demonstrated in vivo and/or in vitro growth capacity . These data suggest that only some tumors, presumably with peculiar biological characteristics, can efficiently grow in these artificial systems.

Biochim Biophys Acta, 1989 Dec 28, 987(2), 239 - 42
Net efflux of chloride from cell suspensions measured with a K+ electrode; Rothstein A et al.; Under appropriate conditions (presence of cation ionophores) net KCl efflux measured with a K+ electrode can be used to estimate conductive Cl- fluxes, a sensitive procedure that allows continuous recording . The procedure was tested in human red cells by demonstrating effects of ionophores and of an anion transport inhibitor, and in dissociated MDCK cells by demonstration of cAMP and volume-activated Cl- fluxes.

Biochemistry, 1989 Dec 26, 28(26), 10061 - 5
Coenzyme F430 as a possible catalyst for the reductive dehalogenation of chlorinated C1 hydrocarbons in methanogenic bacteria; Krone UE et al.; Corrinoids, such as aquocobalamin, methylcobalamin, and (cyanoaquo)cobinamide, catalyze the reductive dehalogenation of CCl4 with titanium(III) citrate as the electron donor {Krone et al . (1989) Biochemistry 28, 4908-4914} . We report here that this reaction is also effectively mediated by the nickel-containing porphinoid, coenzyme F430, found in methanogenic bacteria . Chloroform, methylene chloride, methyl chloride, and methane were detected as intermediates and products . Ethane was formed in trace amounts, and several as yet unidentified nonvolatile compounds were also generated . The rate of dehalogenation decreased in the series of CCl4, CHCl3, and CH2Cl2 . With coenzyme F430 as the catalyst, the reduction of CH3Cl to CH4 proceeded more than 50 times faster than with aquocobalamin . Cell suspensions of Methanosarcina barkeri were found to catalyze the reductive dehalogenation of CCl4 with CO as the electron donor (E'0 = -0.524 V) . Methylene chloride was the main end product . The kinetics of CHCl3 and CH2Cl2 formation from CCl4 were similar to those with coenzyme F430 or aquocobalamin as catalysts and titanium(III) citrate as the reductant.

Int J Cancer, 1989 Dec 15, 44(6), 959 - 64
Cytogenetic abnormalities in benign lymphoid hyperplasia: a dual-parameter study using chromosome analysis and flow cytometry; Grace J et al.; This is a prospective study of lymphoid tissue showing benign reactive hyperplasia (18 lymph nodes and 2 tonsils), using cytogenetic analysis of cells stimulated with T- or B-cell mitogens . The reason for this study was the detection of an abnormal chromosomal population in cells from an enlarged lymph node excised from a 7-year-old female who on further investigation was found to be clinically well and after one year's close follow-up had not developed any further signs or symptoms of malignancy . In addition, DNA content was measured by flow cytometry (FCM) on fresh cell suspensions in 17 cases and fixed cell suspensions in 3 cases . Structural and numerical clonal chromosome abnormalities were found in 9 of the 20 samples, but no common specific defect was identified . FCM showed an abnormal DNA content in 10 of the 20 samples studied; 3 of these showed clonal chromosome abnormalities . Surface membrane immunoglobulin studies were carried out on 15 of the 20 samples using cell suspensions and frozen tissue sections . In 5 of the 15 cases, monoclonal surface immunoglobulin was detected . There was no direct correlation between the surface membrane immunoglobulin studies and the chromosome and FCM analyses . We conclude that aneuploidy is a common feature in reactive lymphoid tissue, but both cytogenetics and FCM are needed to identify it.

J Biol Chem, 1989 Dec 5, 264(34), 20502 - 8
Transglutaminase-mediated cross-linking of fibrinogen by human umbilical vein endothelial cells; Martinez J et al.; The interaction of endothelial cells with soluble or substrate-immobilized 125I-labeled fibrinogen (125I-FGN) was analyzed . Binding experiments involved incubation of 125I-FGN with cell suspensions at 4 degrees C . Bound ligand was quantitated by centrifugation of cells through silicone oil followed by scintillation analysis of the cell pellet . Calcium-dependent binding of 125I-FGN reached a maximum after 3 h and represented about 60% of the total . Half-maximal saturation occurred at 60 nM, and about 9 x 10(4) molecules were bound/cell at saturation (approximately 100 nM) . Calcium-dependent binding was completely inhibited by unlabeled fibrinogen, partially inhibited by a monoclonal antibody (7E3) against glycoprotein IIb-IIIa, but not inhibited by fibrinogen fragments D or E, an anti-glycoprotein IIIa polyclonal antibody, or the Arg-Gly-Asp-Ser tetrapeptide . In contrast, the Arg-Gly-Asp-Ser tetrapeptide as well as the monoclonal antibody 7E3 markedly inhibited attachment of endothelial cells to substrate-immobilized fibrinogen, whereas fragment D or E did not . Both in suspension and monolayer, the 125I-FGN underwent cross-linking involving principally the A alpha chain . The transglutaminase inhibitors putrescine, histamine, and cystamine interfered with 125I-FGN binding and cross-linking by suspended cells . Since cross-linking in suspension was limited to bound 125I-FGN and since transglutaminase activity was not detectable in the binding buffer, cross-linking may have been mediated by a cell-associated transglutaminase.

Blood, 1989 Dec, 74(8), 2755 - 63
An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse; Ploemacher RE et al.; We have developed a limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse using a miniturized stroma-dependent bone marrow culture assay in vitro . The cells were overlaid on irradiated stromal layers in microtiter wells in a range of concentrations, and frequencies of cobblestone area-forming cells (CAFC) were calculated by employing Poisson statistics . The production of secondary granulocyte/macrophage colony-forming units (CFU-G/M) in the adherent layer of individual wells was correlated with the presence of such cobblestone areas . CAFC frequencies were determined in bone marrow cell suspensions that were either enriched for marrow repopulating ability (MRA) in vivo, while depleted for spleen colony-forming units (CFU-S), or vice versa . The separation of bone marrow cells (BMC) was either based on centrifugal elutriation, or monoclonal antibody-mediated magnetic depletion of cells carrying cell surface differentiation antigens, and subsequent sorting on the basis of light scatter and rhodamine-123 retention as a measure of mitochondrial activity . In addition, 5-fluorouracil-resistant BMC were studied . Our investigations show that a time-dependent cobblestone area formation exists that reflects the turnover time and primitiveness of CAFC . The frequency of precursors forming cobblestone areas on day 28 after overlay is proposed to be a measure for MRA, whereas the day-7 CAFC frequency closely corresponds with day-12 CFU-S numbers in the suspensions tested.

Blood, 1989 Dec, 74(8), 2624 - 8
Immunophenotypes of Reed-Sternberg cells: a study of 19 cases of Hodgkin's disease in plastic-embedded sections; Casey TT et al.; The immunophenotype of Reed-Sternberg (RS) cells in Hodgkin's disease (HD) has not been clearly defined, partly owing to difficulties in studying RS cells in cell suspensions or identifying them with certainty in frozen sections . We studied the immunophenotype of RS cells with a recently developed plastic section immunohistochemical technique on acetone-fixed tissues that affords superior morphological detail while preserving a wide variety of lymphoid differentiation antigens . Nineteen cases of HD {16 nodular sclerosing (NS), 2 mixed cellularity (MC), and 1 lymphocyte depleted (LD)} were embedded in plastic and stained for pan-B, pan-T, and various T-subset markers, as well as leukocyte common antigen (CD45), interleukin-2 (IL-2) receptor (CD25), and RS cell markers CD15 and CD30 . RS cells were positive for CD45, CD15, CD30, and CD25, except for 3 cases (2 NS, 1 MC) that were CD15 negative and 2 cases (NS) that were CD45 negative . In 10 cases (NS), RS cells were positive for at least two pan-T-cell markers and CD4; pan-B cell markers were uniformly negative . RS cells in 6 cases (3 NS, 2 MC, 1 LD) were positive for at least one T-cell marker (CD2) and one B-cell marker (CD22) . Two cases of NSHD showed no T- or B-cell marking . These data provide further evidence that RS cells in some cases of NSHD have T-cell phenotypes and that RS cells are not homogeneous in their immunoreactivity.

Gematol Transfuziol, 1989 Dec, 34(12), 9 - 12
{A comparative evaluation of the effect of a number of crystalloid and colloid solutions on the rheological properties of blood}; Voitenko II; Dynamic viscosity of the blood (DVB) and red blood cell deformability were studied in 10 donors after in vitro addition to the blood of crystalloid and colloid solutions which are used in the clinical practice for transfusion therapy . Investigations were conducted with the use of Geppler's viscosimeter and filtration of red blood cell suspension . A conclusion has been made that crystalloid solutions are more preferable than colloid solutions for DVB reduction and improvement of red blood cell deformability.

Mol Cell Endocrinol, 1989 Dec, 67(2-3), 179 - 84
Morphometric analysis of corpus allatum cells in adult females of three cockroach species; Chiang AS et al.; The number of cells and their sizes in the corpus allatum (CA) of adult female Blattella germanica, Supella longipalpa and Diploptera punctata were determined during oocyte maturation . Cell number and size were directly measured in cell suspensions following enzymatic dissociation of freshly excised CA . Cell numbers were verified by total cell counts in whole-mount CA monolayers and by hemocytometric sampling . In all three species, cell number did not change during the period of CA activation, averaging ca . 2000 cells per gland in B . germanica, 3500 cells per gland in S . longipalpa and 11,000 cells per gland in D . punctata . Cell diameter increased significantly in all three species during this period from a mean value of 8.9 microns to 11.7 microns in B . germanica, from 9.2 microns to 14.6 microns in S . longipalpa and from 10.0 microns to 15.6 microns in D . punctata . During a 4 h incubation period, dissociated CA cells incorporated L-{methyl-3H}-methionine into juvenile hormone-III at rates comparable to intact glands . These data suggest that CA activation in the first ovarian cycle of these species is associated mainly with an increase in cell size with minor changes in cell number.

Ecotoxicol Environ Saf, 1989 Dec, 18(3), 337 - 45
Uncoupling properties of a chlorophenol series on Acer cell suspensions: a QSAR study; Ravanel P et al.; A series of 22 chlorinated phenols was investigated for their uncoupling effect on Acer cell suspensions . All the studied molecules were able to enter the cell and had instantaneous uncoupling properties: pentachlorophenol was the best uncoupler of the series (the concentration required to uncouple by 50% was 5 microM), being 200-fold more effective than 3-Cl or 2-Cl phenol, which were the molecules having the lowest activity . The QSAR (quantitative structure-activity relationship) study gave good equations for the uncoupling activity with the steric and electronic parameters taken together . The electronic parameter sigma was always present with a positive sign, whereas A, the angular parameter, was always negative . The general steric parameters (sigma D, 1 chi v, MR) represented the third component of the equations and each of them played practically an equivalent role . Equations of quality (r greater than 0.9), which were statistically significant, could be obtained after four compounds in F (2,5-diCl phenol; 2,4,5-triCl phenol; 2,4-diCl 6-NO2 phenol; and 2-Cl 4,6-diNO2 phenol) had been excluded and when quadratic terms were not present . The best equation (r = 0.940) was obtained with sigma D, A, and sigma 1 . The comparison of the significant equations with those previously established with isolated mitochondria led us to suppose that there were no selective limiting steps during the transfer of the studied compounds from the reaction medium to mitochondria inside the cells . The major difference between the uncoupling results obtained with isolated mitochondria and with cells was that the concentrations needed to uncouple by 50% were always higher in cells.

J Appl Physiol, 1989 Dec, 67(6), 2593 - 9
Reduced erythrocyte deformability alters pulmonary hemodynamics; Doyle MP et al.; Isolated rat lungs were perfused with suspensions containing normal and stiffened erythrocytes (RBCs) to assess the effect of altered RBC deformability on pulmonary hemodynamics . RBC suspensions were prepared using cells previously incubated in isosmolar phosphate-buffered saline with or without 0.0125 or 0.01875% glutaraldehyde . Washed RBCs were resuspended in isosmolar 4% albumin saline solution . Isolated rat lungs were perfused with control and stiffened cells by the use of a perfusion system that allowed rapid switching between suspensions . Pressure-flow (P/Q) curves were constructed by measuring pulmonary arterial pressure (Ppa) over a range of flow rates . In a second set of experiments, P/Q curves were generated for perfusion with control and stiffened cells (0.0125% glutaraldehyde) before and after vasoconstriction with a synthetic prostaglandin analogue (U 46619) . RBC deformability was quantified in all experiments by determination of filtration time of a dilute cell suspension through a 4.7 microns Nuclepore filter . Incubation with 0.0125 or 0.01875% glutaraldehyde produced a 6 or 21% decrease in RBC deformability, respectively . These decreases in deformability were associated with significant increases in Ppa at each flow rate . The increases in Ppa correlated significantly with the degree of RBC stiffening . With 0.0125% glutaraldehyde, the P/Q curve was shifted upward without a change in slope, whereas incubation with 0.01875% glutaraldehyde resulted in a significant increase in slope . Vasoconstriction and perfusion with stiffened RBCs had additive effects on Ppa . These findings suggest that decreases in RBC deformability cause physiologically significant elevations in hemodynamic resistance in the pulmonary circuit independent of vasoactivity.

Anal Quant Cytol Histol, 1989 Dec, 11(6), 384 - 90
Influence of the elimination of lymphocytes from tumor cell suspensions on the calculation of S-phase fractions by flow cytometry; Eckhardt R et al.; Contaminating lymphocytes were eliminated from enzymatically obtained cell suspensions of 260 surgical biopsy specimens by density gradient centrifugation using lymphocyte separation medium (LSM) in an attempt to improve the determination of S-phase fractions by flow cytometry . The elimination of lymphocytes from the cell suspensions was ascertained on cytologic smears prepared from the suspensions before and after LSM centrifugation . Following the elimination of lymphocytes, the calculated S-phase fractions increased significantly in DNA-diploid tumors, but not in DNA-aneuploid ones . The increase of the S-phase fraction was correlated to the numbers of lymphocytes in the tumor cell suspensions before LSM centrifugation . Furthermore, in 12 tumors originally classified as diploid, an aneuploid cell line was detected after LSM centrifugation . These results indicate that samples from diploid tumors containing large numbers of lymphocytes should have the lymphocytes eliminated by methods such as LSM centrifugation in order to obtain reliable results for the calculations of the S-phase fractions.

Cancer Res, 1989 Dec 1, 49(23), 6840 - 4
Flow cytometric determination of the frequency and heterogeneity of expression of human melanoma-associated antigens; Berd D et al.; We used flow cytometry to measure the expression of human melanoma antigens on cell suspensions dissociated from metastatic masses . The objective was to study the heterogeneity between tumor samples from different patients and between different tumors excised from a single patient . Fifty-three metastases excised from 34 melanoma patients were analyzed with a panel of nine murine monoclonal antibodies (MOABs) . Melanoma cells were stained by an indirect fluorescent method and analyzed on a Coulter EPICS C flow cytometer after gating to exclude tumor-infiltrating leukocytes and dead cells . The most consistently and most strongly expressed antigen was the high-molecular-weight proteoglycan (detected by the MOAB 9.2.27), which was expressed on 95% of the melanoma specimens and by a high proportion of cells within each specimen (mean +/- SE, 79.2 +/- 5.5) . However, strong expression of this antigen was limited to melanoma cells that had been dissociated mechanically and was markedly diminished by exposure to collagenase . Culture of collagenase-dissociated tumor cells for 24 to 48 h resulted in reexpression of the antigen . The expression of other melanoma-associated antigens was not affected by collagenase treatment, but for these antigens there was more variability between cells from an individual tumor and between tumors from different patients . The percentage of enzyme-dissociated tumors considered positive for MOAB binding (defined as at least 10% of cells positive) and the mean +/- SE of the percentage of positive cells within a tumor were as follows: MOAB ME-9-61 (antigen, p97) = 84% + (41.2 +/- 5.4%); MOAB ME-20.4 (antigen, nerve growth factor receptor) = 40% + (18.7 +/- 5.1%); MOAB ME-24 (antigen, ganglioside GD3) = 84% + (50.8 +/- 4.8%); MOAB ME-311 (antigen, ganglioside 9-O-acetyl-GD3) = 76% + (42.5 +/- 5.1%); MOAB ME-361 (antigen, mainly ganglioside GD2) = 3% + (1.9 +/- 0.8%); MOAB 3F8 (antigen, ganglioside GD2) = 36% (10.5 +/- 3.8%); MOAB 14G2a (antigen, ganglioside GD2) = 86% + (46.0 +/- 6.7%); MOAB L243 (antigen, HLA-DR) = 56% + (22.5 +/- 5.5%) . In 19 cases, we were able to compare the antigenic profiles of two tumors excised from the same patient at different times . Analysis by nonindependent t test showed no significant differences in MOAB binding between the paired tumors . Moreover, linear regression analysis indicated that there was a linear relationship, with a slope approximately = 1, between the percentage of positive cells in Tumor 1 versus Tumor 2.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Struct Funct, 1989 Dec, 14(6), 669 - 72
Dual-fluorescence flow cytometric analysis of membrane potential and cytoplasmic free Ca2+ concentration in embryonic rat hippocampal cells; Mitsumoto Y et al.; We have demonstrated simultaneous measurement of the membrane potential and cytoplasmic free Ca2+ concentration ({Ca2+}i) by utilizing a dual-laser flow cytometer in embryonic rat hippocampal cell suspensions . Veratrine, a Na+ channel activator, induced both membrane depolarization and elevation of {Ca2+}i . These actions of veratrine were all reversed by the presence of tetrodotoxin (TTX) . These findings suggest that Na+ channels are functionally expressed in the cells and the activation of Na+ channels increases {Ca2+}i . The usefulness of the flow cytometric analysis in elucidating the expression of membrane functions in the embryonic central nervous systems (CNS) is discussed.

J Immunol, 1989 Dec 1, 143(11), 3659 - 65
Release of platelet-activating factor and the metabolism of leukotriene B4 by the human neutrophil when studied in a cell superfusion model; Cluzel M et al.; Stimulated human neutrophils are known to synthesize large quantities of 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) and 5,12-dihydroxy-6,14-cis-8,10-trans-transeicosatetraenoic acid (LTB4) . However, in an isolated cell suspension the majority of synthesized PAF appears to remain cell associated . In addition, LTB4 is rapidly metabolized to an omega-oxidation product (20-OH-LTB4) . Experiments were designed to test the hypothesis that the degree of association of PAF with the neutrophils and the metabolism of LTB4 by the neutrophils is a result of the in vitro condition used during cell activation . Here we have compared in paired experiments ionophore A23187-induced production of PAF and LTB4 by human neutrophils in a concentrated cell suspension, a diluted cell suspension and in a system in which the cells are placed on a matrix and superfused with buffer at a constant flow rate (dynamic system) . There was little difference in the amount of PAF synthesized in the concentrated cell suspension and the dynamic system . However, less PAF was produced by neutrophils in the dilution system . The percent of PAF released was consistently greater in the dynamic and dilution systems than in the concentrated cell suspension . For example, more than 40% of PAF measured by incorporation of {3H}acetate or gas chromatography/mass spectrometry was released in the dynamic system and dilution systems . In contrast, less than 15% of the PAF synthesized was released from the cells in the concentrated cell suspension . 1-0-Hexadecyl-2-acetyl-3-GPC was primarily released from the neutrophils . By contrast both 1-0-hexadecyl and 1-0-octadecyl linked species of PAF were found within the cells . Exogenous PAF added to neutrophils in the dynamic or dilution systems was taken up and metabolized at a significantly lower rate than that added to cells in the concentrated cell suspension . Most of the leukotrienes synthesized by the neutrophil during A23187 stimulation were released from the cells . However, studies of LTB4 metabolism revealed differences between the dynamic and concentrated cell suspension designs . By 20 min, most of the LTB4 was recovered as 20-OH-LTB4 in the concentrated cell suspension, whereas in the dynamic system little 20-OH-LTB4 was found in the superfusate over 20 min . These experiments suggest that a large proportion of PAF synthesized by neutrophils may be released . They also suggest that the omega-hydroxylation of LTB4 by neutrophils occurs after synthesized LTB4 is released and taken back up by the cell.

J Neurochem, 1989 Dec, 53(6), 1952 - 4
A kainate receptor linked to nitric oxide synthesis from arginine; Garthwaite J et al.; In slices of young rat cerebellum, the glutamate analogue kainate induced a large accumulation of cyclic GMP, which was inhibited by non-N-methyl-D-aspartate antagonists . Quisqualate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate evoked only small cyclic GMP responses and inhibited the effect of kainate . When tested in cerebellar cell suspensions, glutamate was also a potent antagonist of the cyclic GMP response to kainate . Superoxide dismutase enhanced the response in the isolated cells, whereas haemoglobin and methylene blue were inhibitory . The response in slices was Ca2+ dependent, augmented by arginine, and inhibited by L-NG-monomethylarginine in a manner that could be reversed by additional arginine . It is concluded that stimulation of kainate receptors leads to activation of the enzyme that synthesizes nitric oxide from arginine and that activation of soluble guanylate cyclase by the released nitric oxide accounts for the cyclic GMP generation.

Rinsho Ketsueki, 1989 Dec, 30(12), 2134 - 40
{Flow cytometric analysis of surface phenotypes in B-cell non-Hodgkin's lymphoma}; Chubachi A; The objective of this study is to demonstrate the diagnostic usefulness of flow cytometric analysis of surface immunoglobulin (S-Ig) light chains and monoclonal antibodies (MoAbs) in B-cell non-Hodgkin's lymphoma . For this purpose, the biopsied specimen (lymph nodes, tonsils, and spleens etc) cell suspensions from 44 patients were studied to detect the expression of S-Ig and several antigens recognized by MoAbs . A tumor was considered B-lineage if expression of pan B antigens and/or monoclonal light chains (S-Ig and/or cytoplasmic Ig) was detected in the absence of pan T antigens . Monoclonal expression of S-Ig light chain was detectable in 37 of 44 (84%) patients with B-cell lymphoma . In two of three cases having polyclonal S-Ig light chain, monoclonal C-Ig light chain was detected in large cells on paraffin-embedded tissues with immunohistochemical technique . Pan B antigens were strongly positive in four cases with negative S-Ig light chain . In diffuse large cell lymphoma, expression of CD11b was restricted to non-cleaved cell type (DLN) . CD10 was favorably expressed by non-cleaved cell type and to a lesser extent, by immunoblastic histology (LI), but was not detected on cases with cleaved cell type (DLC) . Furthermore, relatively uniform expression of S-IgM was seen in DLC subgroup, and on the contrary, heterogenous S-Ig was shown in other histology groups (DLN, LI) . Flow cytometry provides a rapid, objective technology to confirm the immunological diagnosis of B-cell non-Hodgkin's lymphoma.

J Vasc Surg, 1989 Dec, 10(6), 650 - 5
The potential unreliability of indium 111 oxine labeling in studies of endothelial cell kinetics; Patterson RB et al.; The current widespread use of indium 111 oxine for labeling endothelial cells to study their interaction with various bioprosthetic flow surfaces presupposes a high retention of the radioisotope within the cell and a lack of significant adherence of any marker released from the cell to the surface under study . We measured the loss of indium 111 from freshly harvested, canine, venous endothelial cells, and their viability in cell culture, for varying intervals up to 24 hours . At prescribed intervals, aliquots of the radiolabeled endothelial cell suspension were centrifuged, and the supernatant was separated from the cell pellet . The relative radioactivity of each was measured in a gamma well counter and the spontaneous loss of marker from the endothelial cells was calculated . Spontaneous loss of indium 111 was 7.8% +/- 1.9% at 1 hour and 47.6% +/- 1.8% at 24 hours . Loss of activity was virtually constant between 14 and 24 hours . Endothelial cell viability was 80% at 24 hours . We next studied the in vitro affinity of indium 111 oxine and indium 111 transferrin for untreated expanded polytetrafluoroethylene vascular grafts and for similar grafts treated with two surfactants commonly used to increase the "wetability" of expanded polytetrafluoroethylene, a nonionic sufectant (Nonidet) and tridodecylmethylammonium chloride, and with two glycoproteins, fibronectin and basement membrane gel . A minimum of three graft segments were studied in each group . The affinity of graft material for indium 111, in both its oxine and transferrin complex, was significantly increased by treating the graft with a wetting agent, and it was further increased by the addition of a glycoprotein.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Biophys, 1989 Dec, 15(3), 159 - 71
Use of biocarrier beads and flow cytometry for single-cell studies of fibronectin gene regulation in dibutyryl cyclic AMP reverse transformed CHO-K1 cells; Sterner JM et al.; The protease sensitivity of a number of cell surface or cytoskeletal components and the relationship of these to the substratum in attached cells has prevented the quantitative measurement of their expression by flow cytometry . Using traditional cell sorting techniques, cells must be treated with a protease to detach them from a substrate in order to produce a single-cell suspension . Unfortunately, proteolytic treatment alters or destroys a number of cellular proteins . Fibronectin either on the cell surface or as part of the substratum laid down by the cell is particularly sensitive to proteases, preventing its quantitative study by flow cytometry . To circumvent these problems and produce a single cell suspension necessary for flow cytometric analysis, CHO-K1, a Chinese hamster ovary cell line, were grown in suspension on specially-treated 25 microns biocarrier beads . The CHO-K1 cell line is composed of transformed epithelial-like cells that have lost the fibronectin deposit around their cell membranes . To restore the typical fibroblastic deposit of fibronectin, the cells attached to beads were induced by dibutyryl cAMP to undergo a reverse transformation reaction to restore fibroblastic morphology and the typical fibroblastic deposite of fibronectin on the cell surface and substratum . The cells attached to beads were then immunofluorescently labeled for the protease-sensitive, extracellular matrix component, fibronectin, and examined on a flow cytometer . Cell surface fibronectin heterogeneity was then examined on a cell-by-cell basis as a function of cell cycle using Hoechst 33342 dye that binds to AT base pairs of cellular DNA . Dual laser measurement and multiparameter list mode data analysis were used to study the relationship between cell surface fibronectin of biocarrier bead attached cells and cell cycle.

Brain Res, 1989 Nov 27, 503(1), 111 - 7
Myelination of axons within cytosine arabinoside treated mouse cerebellar explants by cultured rat oligodendrocytes; Seil FJ et al.; Cell suspensions of cultured purified rat oligodendrocytes prepared by the differential substrate adhesion method were applied to neonatal mouse cerebellar explant cultures in which myelination and oligodendrocyte maturation had been irreversibly inhibited by exposure to cytosine arabinoside . Myelination of Purkinje cell axons within 92% of the host explants was observed 2-5 days after oligodendrocyte application . Ultrastructurally, mature oligodendrocytes and axons surrounded by compact myelin, as well as spherules of compact myelin membranes without axons, were present within the cerebellar explants . It is evident that cultured dissociated purified oligodendrocytes retain the ability to myelinate appropriate axons . Such oligodendrocytes may be hyperreactive with regard to myelin membrane formation, as suggested by the presence of spheres of compact myelin without axons.

Neurosci Lett, 1989 Nov 20, 106(1-2), 141 - 6
Functional expression of a subtype of the Ca2+ channels in the embryonic rat hippocampus as detected by dual-fluorescence flow cytometric analysis; Mitsumoto Y et al.; We have analyzed the expression of the Ca2+ channels in hippocampal cell suspensions from the 18- to 20-day-old rat embryo using dual-fluorescence flow cytometry . A high concentration of K+ induced elevation of the cytoplasmic free Ca2+ concentration ({Ca2+}i) as well as membrane depolarization . The high K+-evoked {Ca2+}i increase was inhibited by phenytoin, but not by either nifedipine or nicardipine . These agents had no effect on the high K+-induced membrane depolarization . These findings suggest that a subtype corresponding to the low voltage-activated Ca2+ channel is expressed in the embryonic rat hippocampal cells.

Ultrasonics, 1989 Nov, 27(6), 362 - 9
Confirmation of the protective effect of cysteamine in in vitro ultrasound exposures; Inoue M et al.; Armour and Corry (Radiat . Res . (1982) 89 369-380) reported that ultrasound-induced damage to in vitro Chinese hamster ovary cells was significantly reduced in the presence of cysteamine . The objective of this study was to attempt verification of this result . Four series of experiments were undertaken using in vitro cell suspensions, namely: (1) determination of the effect of cysteamine concentration on cell growth; (2) determination of the temperature dependence of ultrasonically induced cell damage; (3) determination of a dose-response relationship for the cytotoxicity of cysteamine; and (4) assessment of cell integrity and reproductive capacity in the presence or absence of cysteamine during ultrasonic exposure . Ultrasound parameters included a resonance frequency of 1 MHz, a continuous wave exposure duration of 5 min, and intensities from 0 to 21.6 W cm-2 . The results indicated a dependence of ultrasound's efficacy on the medium's temperature during insonation and a significant reduction of ultrasound efficacy in compromising cellular integrity in the presence of cysteamine.

Int J Cell Cloning, 1989 Nov, 7(6), 385 - 94
Antitumor activity of hyperthermia alone or in combination with cisplatin and melphalan in primary cultures of human malignant melanoma; Zaffaroni N et al.; The effects of heat and the interaction between hyperthermia and alkylating agents, such as cisplatin (CDDP) and melphalan (L-PAM) in human malignant melanoma biopsies have been investigated by a short-term assay based upon the inhibition of 3H-thymidine incorporation . Cell suspensions from 50 cutaneous and lymph nodal metastases were heated at 40.5 degrees C or at 42 degrees C for 1 h . There were significant antiproliferative effects due to heat in 10% of the tumors exposed to 40.5 degrees C and 34% to 42 degrees C . Thermal resistance was evident in 73% (at 40.5 degrees C) and 54% (at 43 degrees C) of tumors, and there was significant enhancement of cell growth in 17% and 12% of tumors . The combined effects of hyperthermia and drugs were studied on 36 tumors . Cell suspensions were exposed to different concentrations of CDDP or L-PAM for 1 h at 40.5 degrees C and 42 degrees C . Synergy between heat and CDDP was observed in 7% of cases treated with the lowest drug dose and 38% of cases treated with the highest (40.5 degrees C), with only a slight increase in the frequency of synergy at 42 degrees C . Synergy between heat and L-PAM was also observed in 12% to 44% of tumors at 42 degrees C as a function of drug concentration.

J Histochem Cytochem, 1989 Nov, 37(11), 1653 - 8
Use of a FACS III for fluorescence depolarization with DPH; Bock G et al.; We have previously demonstrated age-related differences in human lymphocyte membrane fluidity, by use of steady-state polarization measurements on bulk cell suspensions with the fluorescence probe DPH . However, for exact analysis of the possible functional importance of these changes, single-cell measurements were deemed of interest . We have now used an analog division device to measure fluorescence depolarization "p" of DPH in real time with a FACS III flow cytometer . The measurements are reliable, as we have been able to confirm the differences in DPH "p" between monocytes and lymphocytes previously shown in bulk suspension and to demonstrate the expected differences in fluidity of lipid-modulated cells . We also found significant differences in DPH "p" between lymphocytes of young and elderly blood donors . Lymphocyte subsets did not differ in polarization values but did differ in fluorescence intensity with Th less than Ts less than B = NK cells.

Diabetes Res, 1989 Nov, 12(3), 141 - 9
A microfluorometric viability assay for isolated human and rat islets of Langerhans; London NJ et al.; A microfluorometric viability assay for isolated human and rat islets of Langerhans has been developed using the fluorochromes fluorescein diacetate and propidium iodide . Fluoroscein diacetate causes live cells to fluoresce green under blue light excitation (490 nm); propidium iodide causes dead cells to fluoresce red under green light excitation (545 nm) . The fluorescence intensity from the live and dead cells within a single islet was selectively measured by photometry using 520 nm and 610 nm barrier filters with blue and green light excitation respectively . All measurements were corrected for background fluorescence . It was necessary to incubate single islets with the fluorochrome mixture for 105 min in order to achieve maximum fluorescence intensity . It was found that when 50 microliters of a fluorochrome mixture containing 0.67 mumol/l fluorescein diacetate and 4.0 mumol/l propidium iodide was incubated with a single islet and the fluorescence from live (blue light excitation) and dead (green light excitation) cells measured, then the proportion of dead cells within the islet was equal to the (propidium iodide fluorescence)--(0.04 x fluorescein fluorescence) divided by the sum of the (fluorescein fluorescence) and (propidium iodide fluorescence--(0.04 x fluorescein fluorescence} . The proportion of dead cells within single human or rat islets measured by microfluorometry was found to correlate highly significantly (r = 0.99, p less than 0.001) with the proportion of dead cells measured by dissociating the same islet into a single cell suspension and counting the actual proportion of dead cells . This assay therefore provides a rapid, accurate and objective measurement of the proportion of dead cells within isolated human and rat islets.

Mol Cell Biol, 1989 Nov, 9(11), 4819 - 23
Characterization of phytochrome-regulated gene expression in a photoautotrophic cell suspension: possible role for calmodulin; Lam E et al.; A photoautotrophic suspension culture of soybeans was found to exhibit light-dependent expression of the genes encoding the major chlorophyll a- and b-binding protein (CAB) . The expression was mediated by phytochrome, since it was induced by red light and reversed by far-red light . The maximal level as well as the kinetics of the induction were comparable between the suspension culture and soybean seedlings . Using this cell culture, we addressed the question of whether a calcium- and/or calmodulin-mediated step is involved in the signal transduction process between phytochrome and CAB expression . We found that W-7, a potent calmodulin antagonist, severely attenuated the induction of CAB mRNA by light, whereas W-5, a weak calmodulin antagonist, had little effect . Control experiments demonstrated that W-7 treatment did not block the induction of hsp-75 by heat shock . The addition of ionomycin, a calcium ionophore, induced a low level of CAB mRNA accumulation in the dark which could be further enhanced by light treatment . We propose that calmodulin activation by light is necessary but not sufficient to induce maximal CAB expression.

Am J Pathol, 1989 Nov, 135(5), 889 - 97
Immunoreactivity for IL-1 beta and TNF alpha in human lymphoid and nonlymphoid tissues; Ruco LP et al.; Monoclonal antibodies (MAbs) against two non-cross-reacting antigens of human IL-1 beta (Vhp20 and BRhC3) and human TNF alpha (B154.2 and B154.7) were applied to identify cytokine-containing cells in tissue sections and in cell suspensions . IL-1 beta- or TNF alpha-positive cells were not present in immunostained cytocentrifuge smears prepared from freshly isolated peripheral blood leukocytes, spleen, and lymph node cells . After 18 hours of culture with bacterial endotoxin (LPS), 80% to 90% of blood monocytes, 30% of spleen macrophages, and 2% to 28% of lymph node macrophages were strongly positive for IL-1 beta with either of the MAbs . Furthermore, 25% to 35% of blood monocytes and 6% to 60% of lymph node macrophages were stained for TNF alpha . Cells positive for IL-1 beta or TNF alpha were extremely rare in sections of normal thymus, spleen, and lymph nodes . Immunoreactivity for IL-1 beta or TNF alpha was frequently observed in sections of granulomatous lymphadenitis (N = 11) . IL-1 beta or TNF alpha staining was confined to the epithelioid macrophages forming the granuloma, and the intensity of TNF alpha reactivity was generally stronger . The high frequency of cytokine-containing cells in this pathologic condition was confirmed in a cell suspension study showing that 20% of epithelioid macrophages were weakly positive for IL-1 beta and 80% were strongly positive for TNF alpha . The presence of cytokine-containing cells was investigated in cryostat sections of several nonlymphoid organs with normal histologic appearance . IL-1 beta reactivity was not observed in any of the tissues . TNF alpha reactivity was frequently demonstrated in isolated macrophages embedded in the interstitial connective tissue.

Endocrinology, 1989 Nov, 125(5), 2636 - 44
Effects of stimulation of adenylate cyclase and protein kinase-C on cultured fetal rat B-cells; Mourmeaux JL et al.; To study the maturation of fetal pancreatic B-cells, cell suspensions of pancreas from 21.5-day-old fetuses were cultured in RPMI medium containing 10 mM glucose . Forskolin (1 microM), used to stimulate adenylate cyclase, moderately delayed the neoformation of islets, slightly accelerated the proliferation of endocrine cells, and considerably increased insulin release by the cultures . The latter increase was not completely compensated for by the stimulation of insulin biosynthesis, so that the islet insulin content was decreased . The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 25 nM), used to stimulate protein kinase-C, had little effect on the evolution of the cultures, but increased insulin release . This increase was almost compensated for by the stimulation of insulin biosynthesis . After 9-10 days of culture, insulin release in response to 15 mM glucose or 10 mM leucine was studied with perifused islets . In control islets, glucose produced a sustained increase in insulin release, which, however, was 6-fold smaller than that produced by leucine . Addition of forskolin or TPA to the perifusion medium markedly amplified the response to glucose without causing a biphasic pattern of release . In islets cultured with forskolin, the insulin response to glucose or leucine was decreased, largely owing to the lower insulin stores . In islets cultured with TPA, the insulin response to glucose or leucine was also decreased, but these differences cannot be explained simply by changes in insulin content . Neither treatment affected the kinetics of release . In conclusion, acute stimulation of adenylate cyclase or protein kinase-C markedly increased insulin release from fetal islets without causing an adult-like biphasic pattern of secretion . Chronic stimulation did not accelerate maturation of B-cells.

Haematologica, 1989 Nov-Dec, 74(6), 541 - 5
Human tumor cells cultured "in vitro" activate platelet function by producing ADP or thrombin; Zucchella M et al.; We studied the effects on platelet function of different human tumour cells cultured "in vitro": Mo T lymphocyte cell line, NCI-N592 small cell lung carcinoma cell line, and 5637 bladder carcinoma cell line . Mo and NCI-N592 cells possessed a slight, dose-dependent platelet aggregating activity, which was completely abolished by apyrase and unaffected by hirudin . The cell-free supernatant also induced an aggregation response, which was very similar to that obtained with tumour cell suspensions . The presence of ADP in the cell-free supernatants of cell suspensions was confirmed by HPLC analysis . On the contrary, aggregation induced by 5637 cells was preceded by a significant lag phase; it was not affected by apyrase but it was abolished by hirudin, and the cell-free supernatant had no effect . These data suggest that Mo and NCI-N592 cells activate platelets by producing ADP, while 5637 cells stimulate platelet function by generating thrombin . The amount of ADP produced by the first two tumour cell lines was measured by bioassay: the extent of such production was similar for both cell lines and the maximum was reached after 60 minutes and maintained for up to 3 hours . These results suggest that neoplastic cells can activate platelets by different mechanisms: such investigations should be performed in homologous systems and in well-defined experimental conditions.

Q J Med, 1989 Nov, 73(271), 1071 - 87
Variable corticosteroid sensitivity of thymic cortex and medullary peripheral-type lymphoid tissue in myasthenia gravis patients: structural and functional effects; Willcox N et al.; The thymus has been studied in myasthenia gravis patients to assess the effects of previous immunosuppression on total yields of cell suspension, immunohistology and culture responses . The reduction in cell yields by pretreatment with corticosteroid was very variable . In 16 of 32 cases, cortical, medullary and total cell numbers were all greatly reduced ('depleted cases'), whereas in the others, they were within or near the typical range for untreated myasthenics . Cortical thymocytes were even more depleted than precursor thymic blasts . Thus the interpatient differences in sensitivity to corticosteroid recently described for mature T cells also affected immature cortical thymocytes and their differentiating medullary progeny . In the medulla, mature (CD3+) T lymphocytes and germinal centres were enriched by the loss of cortex and appeared relatively healthy, but somewhat depopulated . Concomitantly, in-vitro T-cell responses to acetylcholine receptor (AChR) and production of anti-AChR antibody and total IgG by thymic cells were usually well within the typical range (assessed per 10(6) cells) . Moreover, the total productivity of the entire thymus was reduced almost entirely by the cellular depopulation rather than by decreased function per surviving cell . Thus the main actions of this alternate day therapy with corticosteroids were apparently on total peripheral cell numbers, and perhaps on activated cells and effector mechanisms too, and its thymic effects were inessential.

Microvasc Res, 1989 Nov, 38(3), 269 - 85
Prediction of oxygen transport rates in blood flowing in large capillaries; Nair PK et al.; A mathematical model has been developed to predict oxygen transport to and from blood flowing in tubes of the diameter of arterioles and larger (approximately 20 microns and larger) . The resistance to oxygen transport in red cell suspensions is much higher than that of a comparable homogeneous hemoglobin solution . The increased resistance is associated with encapsulation of the hemoglobin in the red cells . Yet, somewhat paradoxically, for large capillaries relatively little resistance is within or in the immediate vicinity of the red cells . The great majority of the resistance is shown to be distributed in the plasma . Predictions of oxygen uptake and release are shown to be in excellent agreement with results of measurements taken on red cell suspensions flowing in capillaries of 27- and 100-microns diameter . The model seems to be the first for oxygen transport in flowing blood that is validated by detailed comparison with experimental results . It is a predictive model in that all parameters in the model are determined from independent measurements or from the literature.

J Neurosci Res, 1989 Nov, 24(3), 384 - 90
Intracerebral transplantation of dissociated central nervous system tissue suspensions: use of phaseolus vulgaris leucoagglutinin as a cell marker; Shigematsu K et al.; Successfully transplanted neurons and their sprouting processes were demonstrated by Phaseolus vulgaris leucoagglutinin (PHA) marking . PHA is transported anterogradely and readily reveals the post-transplantation growth of the neuronal processes . Suspensions of fetal central nervous tissue, prepared by dissociation of embryonic rat brain, were marked with PHA and then transplanted into the striatum of nonimmunosuppressed young adult rats . At various intervals thereafter (1 day, 2 days, 1 week, 2 weeks, 1 month, and 2 months), the animals were sacrificed for histological examination with PHA and tyrosine hydroxylase (TH) immunohistochemistry . Up to 2 weeks after transplantation, PHA immunohistochemistry was capable of demonstrating grafted neurons and their presumably regenerated neuronal processes . However, at periods 1 month or longer after transplantation, PHA immunohistochemistry was unreliable . Thus, the PHA marking method has limitations in terms of its retention period, when applied to the intracerebral transplantation of dissociated cell suspensions . Nevertheless, the method presented here can be utilized to study neuronal regeneration as well as the relationship between transplanted neurons and the host tissue.

Br Heart J, 1989 Nov, 62(5), 329 - 34
Changes in the flow properties of white blood cells after acute myocardial infarction; Nash GB et al.; Because they can obstruct blood vessels and release noxious substances, white blood cells may contribute to the development of tissue ischaemia . The flow properties of white cells were tested after myocardial infarction, by measuring the filtration rates of cell suspensions through 8 microns pore filters . Compared with mononuclear cells from age matched controls, mononuclear cells from patients with infarction showed impaired filterability within the first day after the onset of pain; this condition persisted for at least two days and by day 10 it was improved . On day 1, granulocyte filterability and the proportion showing morphological evidence of activation were nearly normal . By day 3 the flow resistance and activation had increased, but the changes seen depended on the age of the patient . The filterability and activation of granulocytes from patients aged less than 60 were significantly increased from day 1, whereas there were no changes in granulocytes from patients aged greater than 60 years . Suspensions of unfractionated white cells showed changes intermediate between the mononuclear cells and granulocytes . A group of five patients who presented with chest pain but who were subsequently found not to have had an infarction showed no evidence of abnormal filterability or activation . The changes in filterability probably reflect white cell activation, which may have an adverse effect on the perfusion of the ischaemic myocardium.

J Pharm Pharmacol, 1989 Nov, 41(11), 775 - 80
Biocompatibility of wound management products: standardization of and determination of cell growth rate in L929 fibroblast cultures; Turner TD et al.; To facilitate the development of a bioassay procedure by which the biocompatibilities of materials used in wound management may be assessed and compared, those environmental factors affecting cell growth in mouse L929 fibroblast cultures have been identified . Standardization of the initial cell number and frequency of change of medium resulted in the virtual elimination of variation of growth curves of L929 cells cultured in flasks of specified surface area . In addition, three methods for assessing fibroblast growth rate in the presence of alginate products used in wound management were evaluated . These were the haemacytometer counting chamber method, the Coulter counting method, and a liquid scintillation counting method . The first two methods determine the number of cells in a given volume of a cell suspension, whereas the third method determines the rate of synthesis of deoxyribonucleic acid (DNA), and hence cell growth, by measuring the incorporation of {3H}thymidine . The haemacytometer method had significant advantages over the other two procedures in providing both qualitative and quantitative data on culture morphology and cell growth response.

J Clin Lab Immunol, 1989 Nov, 30(3), 131 - 4
Effector-target co-aggregation as a crucial step in the neutrophil-mediated tumour cell lysis; Dallegri F et al.; The Daudi cell lysis by human neutrophils, incubated with phorbol myristate acetate (PMA), was inhibited by amino acids (taurine, methionine), consistent with the involvement of hypoclorous acid (HOCl) in the lytic process . Also, the lysis was inhibited by a monoclonal antibody (mAb J-90) directed against the leukocyte function-associated antigen-1 (LFA-1) . The inhibition of the target cell lysis by mAb J-90 is not due to a HOCl-scavenging mechanism, as suggested by the use of control mAb Dako M-1 (anti CD-15) . As detected by the microscopic examination of samples from test tubes and measured by monitoring the light transmission from the cell suspensions, neutrophils and Daudi cells were found to co-aggregate during the lytic reaction . Co-aggregation was efficiently inhibited by the mAb J-90 . The results suggest that tumour cell lysis by PMA-triggered human neutrophils involves at least two events: production of HOCl and LFA-1-mediated effector-target adhesion.

Endocrinology, 1989 Nov, 125(5), 2486 - 93
Localization, characterization, and quantification of insulin-like growth factor-I-binding sites in the ewe ovary; Monget P et al.; To assess a potential role of insulin-like growth factor-I (IGF-I) in the ewe ovary, the presence of IGF-I receptors and IGF-I-binding proteins was studied by binding assays performed on granulosa cell suspensions, in follicular fluid, and on ovarian sections . On the ovarian sections, labeling was quantified after autoradiography by microphotometry . Competition studies with IGF-I and insulin allowed us to estimate the relative proportions of binding proteins and type I receptors in the different compartments of the ewe ovary . Our results clearly show that saturable, specific, and high affinity IGF-I receptors are present on the ovine granulosa cells . At equilibrium for both granulosa cell suspensions and frozen sections, the Kd value was close to 2 nM . IGF-I binding proteins were also present in follicular fluid and stroma, thecal, and granulosa cells . At equilibrium for follicular fluid, the Kd value was 0.91 +/- 0.27 nM (mean +/- SE) . Moreover, on frozen sections, it was shown that atresia of small follicles (less than 2 mm) was accompanied by a decrease in the number of IGF-I receptors and an increase in the number of IGF-I-binding proteins on granulosa cells . By contrast, this phenomenon was not observed in large follicles . These data indicate that granulosa cells of ewe ovary possess type I receptors, and IGF-I-binding proteins may modulate IGF-I action in the process of follicular growth and atresia.

J Cell Sci, 1989 Nov, 94 ( Pt 3), 545 - 52
A new monoclonal antibody to a cell-surface antigen that distinguishes luminal epithelial and myoepithelial cells in the rat mammary gland; Mahendran RS et al.; A monoclonal antibody (25.5) has been produced that recognises luminal epithelial cells of the rat mammary gland . This antibody together with monoclonal anti-CALLA antibodies, which react with mammary myoepithelial cells, has been used in biochemical, immunocytochemical and flow cytometric studies . Antibody 25.5 bound to proteins of molecular weight 70K and 25K (K = 10(3) Mr) in both the rat milk fat globule membrane and in single cell suspensions prepared from the virgin adult rat mammary gland . Anti-CALLA antibody (J5), recognised a 93-100K protein in the gland extracts, which co-electrophoresed with the CALLA/CD-10 antigen from NALM-6 acute lymphoblastic leukaemia cell line . Antibody 25.5 bound to the luminal surface of rat mammary epithelial cells at all stages of development from neonatal through to pregnancy, lactation and involution . CALLA immunoreactive staining has previously been shown on basally located presumptive myoepithelial cells at all stages of development . Flow cytometric analyses demonstrated that 25.5 and anti-CALLA antibodies stained independent cell populations in suspensions of single cells prepared from purified epithelial elements from the mammary gland of adult virgin rat.

Immunology, 1989 Nov, 68(3), 332 - 40
Application of a novel immunization protocol to the production of monoclonal antibodies specific for macrophages in human placenta; Nash AD et al.; A monolayer depletion/adoptive immunization protocol that biased the immune response towards recognition of placental macrophage (pMO) antigens was established . BALB/c spleen cells immune to human pMO were adsorbed onto monolayers of the B-cell line QIMR-WIL . Monolayer-depleted or unfractionated cells were transferred to irradiated recipients, which subsequently were restimulated with pMO then killed for hybridoma production . Screening of hybridomas revealed an increased proportion of pMO-specific hybridomas following transfer and fusion of monolayer-depleted cells . Two monoclonal antibodies (mAb), L9 and L21, which were generated through application of this protocol, are described . L9 recognized an antigen on cells within the villi in sections of term placenta and freshly isolated pMO . With time in culture, expression of this antigen decreased markedly . Macrophages, but no other cell type, in placental cell suspensions expressed this antigen . L9 failed to react with any peripheral blood cells . Immunoprecipitation and SDS-PAGE analyses indicated that two proteins of molecular weight (MW) 40,000 and 43,000 were recognized by L9 . Sections of term placenta and freshly isolated pMO failed to react with L21 . After 2-3 days in culture, however, most macrophages expressed this antigen . L21 reacted weakly with peripheral monocytes and granulocytes but not other normal peripheral blood cells . Myeloid cell lines reacted strongly with this mAb only after activation with PMA . SDS-PAGE analyses of the L21 immunoprecipitate under non-reducing conditions revealed a single band of 61,000 MW, while two bands of 46,000 and 49,000 MW were detected under reducing conditions . Cellular distribution and molecular weight analyses indicated that the antigens recognized by these two mAb were apparently distinct from previously defined myeloid antigens.

J Clin Invest, 1989 Nov, 84(5), 1446 - 53
A regiospecific monooxygenase with novel stereopreference is the major pathway for arachidonic acid oxygenation in isolated epidermal cells; Holtzman MJ et al.; We investigated the enzymatic mechanisms responsible for AA oxygenation in homogenous cell suspensions obtained by trypsinization of epidermis from healthy subjects . Cell incubation with AA (0.3-150 microM) invariably resulted in the predominant generation of a compound identified as 12-hydroxyeicosatetraenoic acid (12-HETE) by HPLC and by both negative-ion chemical ionization and electron-impact mass spectrometry . Maximal amounts of 12-HETE were 126 +/- 21 pmol/10(6) cells (+/- SE), and concentration-response curves yielded half-maximal levels for 12-HETE similar to PGE2 at 2 microM AA . Two epoxyeicosatrienoic acids derived from AA were also identified . Stereochemical analysis by chiral-phase chromatography demonstrated that the epidermal cell 12-HETE was a mixture of the 12S- and 12R-hydroxy isomers in a molar ratio varying from 2:1 to 8:1 among subjects . Subcellular fractionation into 12,000 g pellet (containing mitochondria) and 100,000 g supernatant (cytosol) and pellet (microsome) demonstrated that greater than 99% of the 12-HETE was generated by enzymatic activity distributed equally in the two pellets . Both mitochondrial and microsomal activities were increased upon addition of NADPH and were inhibited by carbon monoxide, but the molar ratio of 12S/12R-HETE was threefold greater in microsomal than in mitochondrial fractions . The results demonstrate that human epidermis contains active membrane-bound monooxygenase(s) which preferentially generates 12-HETE from AA, exhibits a 12S stereopreference of hydroxylation, and suggests the presence of distinct mitochondrial and microsomal enzyme systems in epidermal cells.

Br J Dermatol, 1989 Nov, 121(5), 577 - 85
Human epidermal basal keratinocytes express CDw29 antigens; Staquet MJ et al.; Two monoclonal antibodies, K20 and 4B4, assigned to the CDw29 cluster of differentiation antigens, were shown to react with basal keratinocytes (BK) . The aim of this study was to identify the antigens recognized by K20 and 4B4 on epidermal cells, and to determine whether they were identical to those found on lymphocytes . Basal keratinocyte-enriched cell suspensions were labelled with 125I and then 1% NP40 cell lysates were used for immunoprecipitation . Under reducing conditions, K20 and 4B4 immunoprecipitated from basal keratinocytes a broad MW 105,000 band and proteins of MW 145,000, 90,000 and 80,000 . Under non-reducing conditions, each band was shifted down by approximately 5000 MW . Metabolic labelling studies demonstrated that the MW 145,000 and 105,000 subunits were synthesized by basal keratinocytes . On lymphoid cells, K20 and 4B4 are known to precipitate glycoprotein complexes made of a broad MW 130,000 protein band (beta subunit) associated with a protein of MW 150,000 (alpha subunit) and proteins of MW 90,000 and 80,000 expressed in very low amounts . The MW 145,000 and 105,000 bands immunoprecipitated by K20 from basal keratinocytes correspond to the alpha and beta subunits present on lymphoid cells . It has recently been demonstrated that K20 recognizes the common beta subunit of the very late antigens family (VLA) and that 4B4 defines the helper-inducer subset of T lymphocytes . The present investigation provides evidence that basal keratinocytes share antigens of the VLA family with lymphoid cells and that they play an important role in the immune response in the skin-immune system.

J Gen Virol, 1989 Nov, 70 ( Pt 11), 2877 - 86
Specificity of the murine T helper cell immune response to various alphaviruses; Mathews JH et al.; We investigated the specificity of the T helper (Th) cell immune response to three alphaviruses: Venezuelan equine encephalomyelitis (VEE), eastern equine encephalitis (EEE) and western equine encephalitis (WEE) . Single cell suspensions were prepared from spleens of virus-primed C3H mice, and T lymphocyte populations were enriched by nylon wool chromatography . T cells were incubated in vitro with irradiated, syngeneic splenic stimulator cells previously exposed to purified virus . Cellular proliferation was measured by {3H}thymidine uptake 5 days post-stimulation . The predominant proliferating cell type secreted interleukin-2 and was of the Th cell phenotype Thy-1+, Lyt-1+,2-, L3T4+ . Stimulation of VEE, EEE and WEE virus-primed Th cells with homologous and heterologous virus resulted primarily in a proliferative response specific for the immunizing virus . The corresponding antibody response, as measured by ELISA using purified virus as antigen, was also specific for the immunizing virus . The magnitude of the blastogenic response of VEE TC-83 virus-primed lymphocytes to a battery of VEE subtype viruses was remarkably similar to schemes of antigenic classification . The results indicate that the dominant Th cell epitopes on these alphaviruses represent regions largely virus-specific and lead to a virus-specific B cell response which does not change over time after primary inoculations of mice with VEE and WEE viruses and multiple inoculations of mice with EEE virus.

Am J Pathol, 1989 Nov, 135(5), 865 - 70
Flow cytometric analysis of epidermal subpopulations from normal and psoriatic skin using monoclonal antibodies against intermediate filaments; van Erp PE et al.; Keratin-type intermediate filament proteins show characteristic expression in normal and pathologic epidermis . Some keratins are restricted to the basal cell layers, and others occur exclusively in the suprabasal compartment . SDS-gel-electrophoresis and immunohistochemistry are generally used for the assessment of keratin profiles and their localizations . In the present investigation, flow cytometric analysis of four different monoclonal antibodies (MAb) against intermediate filament-type proteins, in addition to measurement of relative DNA content, was performed on cell suspensions derived from lesional and clinically uninvolved skin of psoriatic patients and from skin of healthy controls . MAb Ks8.12, reacting with keratins 13 and 16, was used as a marker for hyperproliferation . Pab601 recognizes the basal cell layer(s) of human epidermis . Keratin 10 expression as a marker of keratinization was quantified with RKSE60 and the anti-vimentin MAb MVI was used as a marker for non-keratinocytes . Psoriatic skin showed significantly reduced numbers of RKSE60-positive cells and MVI-positive cells compared with normal skin . In contrast to normal skin and uninvolved skin of psoriatic patients in which only a minority of the cells were Ks8.12 positive, up to 60% of the cell population in psoriatic lesions bound with MAb . Simultaneous measurement of relative DNA content and MAb binding showed that Pab601 binding was associated with cells in S-phase and G2M-phase of the cell cycle, whereas RKSE60 and Ks8.12 binding were associated with diploid cells . Multiparameter flow cytometry allows quantitative population analysis that could lead to a better understanding of the complex mechanisms of epidermal growth control under normal and pathologic conditions.

Br J Pharmacol, 1989 Oct, 98(2), 574 - 80
Antagonism of platelet activating factor-induced chemiluminescence in guinea-pig peritoneal macrophages in differing states of activation; Parnham MJ et al.; 1 . The effects of the platelet activating factor (Paf) antagonists alprazolam, BN 52021, kadsurenone, L 652,731 and SRI 63119 have been studied on Paf-induced chemiluminescence (CL) of guinea-pig, C . parvum-activated peritoneal macrophages in vitro . 2 . All antagonists produced a shift to the right in the dose-response curve to Paf (0.001-10 mumol l-1) . Schild plots for BN 52021, L 652,731, kadsurenone and SRI 63119 were linear, but only for BN 52021 and kadsurenone did the mean slope not differ significantly from unity . Mean pA2 values for BN 52021 and kadsurenone were 6.60 +/- 0.05 and 6.41 +/- 0.14 (mean + s.e.mean) respectively . Calculation of IC50 values for all antagonists (at 0.1 mumol l-1 Paf) gave an order of potency: L 652731 greater than kadsurenone greater than or equal to BN 52021 greater than alprazolam greater than SRI 63119 . 3 . When individual pA2 values for BN 52021 and kadsurenone were plotted against the maximal CL response to Paf of cell suspensions in the absence of antagonist (reflecting the degree of activation of the macrophages by the C . parvum), it was found that the affinity of both antagonists for macrophage Paf receptors remained relatively constant irrespective of the activation state of the cells . 4 . We conclude that activation of guinea-pig peritoneal macrophages does not account for the increased affinity for macrophage Paf receptors previously observed for kadsurenone . Kadsurenone and BN 52021 presumably bind to a site on Paf receptors which is not affected by the activation process, while alprazolam and SRI 63119 are non-specific antagonists . The reason for the difference between the competitive nature of kadsurenone and its structural analogue L 652,731 is unclear.

Immunology, 1989 Oct, 68(2), 215 - 20
Analysis of autoantibodies to glycolipids on thymocytes in New Zealand black mice; Ishii N et al.; Carbohydrate specificities of thymocyte cytotoxic autoantibodies in New Zealand Black (NZB) mice were examined using liposome immune lysis assay (LILA) system . NZB mice progressively produced antibodies to nine neutral glycolipids and two gangliosides tested by 10 months of age . Such increases were not detected in normal BALB/c and CBA/J mice . Among these antibodies, antibodies to ceramide monohexoside (CMH), ceramide dihexoside (CDH), paragloboside (PG), ceramide pentahexoside (CPH), asialo GM1 (GA1), asialo GM2 (GA2), GM4 and GM2 in the plasma of NZB mice were absorbed by single cell suspensions of autologous untreated as well as protease-treated thymocytes . In contrast, antibodies to ceramide trihexoside (CTH), globoside (Glo), and Forssman antigen (For) were not absorbed by these cells . Analysis of glycolipid compositions of young NZB thymocytes demonstrated that strict correlation was observed between the presence of glycolipids on thymocytes and absorbing pattern of anti-glycolipid antibodies by thymocytes . These data indicate that anti-carbohydrate antibodies with thymocyte binding capacity in NZB mice mainly recognize sugar portions of glycolipids rather than glycoproteins on thymocytes . The failure to find such carbohydrate specific antibodies in some normal strains of mice suggested that these antibodies may be important for subsequent immunologic abnormalities in NZB mice.

Proc Natl Acad Sci U S A, 1989 Oct, 86(19), 7527 - 31
Murine epidermal Langerhans cells express significant amounts of class I major histocompatibility complex antigens; Lenz A et al.; Epidermal Langerhans cells (LC) are leukocytes that express major histocompatibility complex (MHC) class II antigens and function as antigen-presenting and accessory cells . Caughman et al . {Caughman, S . W., Sharrow, S . O., Shimada, S., Stephany, D., Mizuochi, T., Rosenberg, A . S., Katz, S . I . & Singer, A . (1986) Proc . Natl . Acad . Sci . USA 83, 7438-7442} reported that LC are deficient in surface expression of MHC class I antigens, implying a specialization of these cells to class II-restricted antigen presentation . To readdress this obviously important issue, we have studied murine epidermal sheets prepared from B6 X BALB/c----B6 bone marrow chimeras 5 months after irradiation and bone marrow reconstitution . This enabled us to distinguish class I of LC from that of surrounding keratinocytes . When sheets were analyzed by immunofluorescence microscopy with monoclonal antibodies specific for donor class I antigens, donor-derived LC but not LC of recipient origin were stained . Appropriate controls for antibody isotype and MHC haplotype were negative . LC in epidermal cell suspensions, prepared from normal BALB/c and BALB/cBy mice (MHC haplotype d), were analyzed by flow cytometry as well as immunofluorescence microscopy . LC were stained by monoclonal antibodies to class I antigens of haplotype d, but not by isotype-matched control antibodies to class I antigens of haplotype k . We also found that LC were virtually depleted from epidermal cell suspensions by treatment with monoclonal antibodies to class I antigens of haplotype d and complement but not by treatment with control monoclonal antibodies and complement . Our data, therefore, show that LC express MHC class I molecules on their surface.

Am J Hematol, 1989 Oct, 32(2), 117 - 22
Hemoglobin Athens-Georgia {alpha 2 beta 2 40(C6)Arg----Lys} in association with beta 0-thalassemia in Tunisia; Mrad A et al.; We describe an Hb Athens-Georgia (Hb A-Ga)/beta 0-thalassemia compound heterozygosity, found in a Tunisian patient . Oxygen binding studies of red cell suspensions of this patient, containing approximately 95% Hb A-Ga, revealed an almost normal oxygen affinity . Nevertheless, dilute solutions of Hb A-Ga showed an increased overall oxygen affinity and decreased heme-heme interaction . This could be explained by a tetrameric hemoglobin with normal oxygen binding properties but with increased dissociation into monomers or dimers, as a consequence of a structural abnormality within the alpha 1 beta 2 interface . Such an interpretation would explain the increased oxygen affinity reported in previous studies performed on heterozygous Hb A/Hb A-Ga patients.

Immunobiology, 1989 Oct, 179(4-5), 432 - 44
Thymocytes reacting with heterologous antibodies against GP 330 in autologous immune complex glomerulopathy (AICG) in the rat . The relation between susceptibility for AICG and anti-GP 330-binding thymocytes; Bagchus WM et al.; Autologous immune complex glomerulopathy (AICG) is induced by immunizing rats with a crude brushborder fraction of rat kidney tubules (Fx1A) or with the purified GP 330 antigen . In these animals, anti-brushborder antibodies develop, leading to subepithelial immune complexes along the glomerular capillary wall . In rats with AICG, thymocytes sensitized against Fx1A as well as thymocytes recognizing anti-Fx1A are present . These latter cells might play a role in the specific tolerance against the pathogenetic antigen GP 330 . To substantiate this notion, immunofluorescence studies were performed in which the number of anti-GP 330 binding cells was quantified in thymus cell suspensions of rats with AICG, in control rats and in naive rats with different genetic background . It is shown that increased numbers of anti-GP 330-binding thymocytes in rats with AICG are associated with a decline in the serum anti-brushborder titer . Furthermore, it appears that the number of anti-GP 330-binding thymocytes in naive rats of the non-responder Brown Norway strain is significantly higher as compared to the PVG/c and Lewis strains, which are susceptible for AICG . The correlation between the numbers of anti-GP 330-binding thymocytes and the susceptibility for AICG suggests a role for these cells in maintaining the tolerance against the Fx1A (GP 330) antigen.

Scand J Immunol, 1989 Oct, 30(4), 399 - 408
In situ study of haemopoiesis in human fetal liver; Kamps WA et al.; The anatomy of haemopoietic cells in human fetal liver was examined using immunohistological techniques on frozen sections of 31 fetuses (10-28 weeks gestational age) . The immunohistological findings were consistent with reported cell suspension data . With regard to the location of haemopoietic activity no particular relationship existed between the various haemopoietic cell lineages . A large number of proliferating cells was present; only a few of these were reactive with haemopoietic progenitor cell monoclonal antibodies (MoAb) CD34 . A population of haemopoietic cells expressed CD43 antigen (MoAb MT1) alone or together with anti-vimentin MoAb reactivity; this population needs further delineation . Erythropoiesis and myelopoiesis occurred in clusters around sinusoids and portal triad vessels respectively . Lack of MoAb reacting exclusively with early developmental stages of erythropoiesis and myelopoiesis precluded dissection of these lineages . Lymphopoiesis occurred in a loosely scattered pattern without any sign of focal development . Pre-B and B-cell numbers increased with gestational age . Cells expressing markers of more mature B cells (surface IgD, CD35, and CD21) were rare . Also, few cells reacted with mature T-cell markers, but CD7+ cells were obviously present . This expression of CD7 on haemopoietic fetal liver cells suggests that T-cell precursors develop in fetal liver as well as B cells.

Boll Soc Ital Biol Sper, 1989 Oct, 65(10), 967 - 73
{New technic for purification of lymphocytes for HLA typing}; Mantovani V et al.; Many of the difficulties encountered in HLA typing derive from the separation of relatively pure cell suspensions for the lymphocytotoxicity test . We have used Immunomagnetic Beads (I.B.) coated with anti-CD8 or CD2 MAb for Class I positive cell selection and I.B . coated with anti-DR for Class II . With this method, compared to traditional techniques, we obtained high purity and viability of cell populations (about 90%), directly from whole blood in 25 minutes . Shorter incubation with antisera and complement was needed and the global time of tissue typing results decreased (150 min . versus 6 hours) . There was no single discrepancy in the tissue typing results between I.B . and traditional methods.

Nippon Hifuka Gakkai Zasshi, 1989 Oct, 99(11), 1145 - 52
{Methods for optimized cultivation of hair cells from C3H mice}; Tanigaki N et al.; Methods for the optimized culturing the hair cells from 4-day-old C3H mice are described . The skin was obtained and soaked in 500 unit/ml dispase at 4 degrees C for 24 hr . Then the epidermis was removed and the dermis was further treated with 0.25% collagenase for 1 hr at 37 degrees C . The hair roots isolated by collagenase digestion of the dermis were dispersed by the treatment with the mixture of trypsin and EDTA into a cell suspension and plated on a substrate . Attachment of hair cells was dependent on serum concentrations and enhanced by collagen coating of the surface of dishes . MCDB 153 was beneficial for the growth of hair cells and discouraged for the growth of dermal fibroblasts . The cells could not grow at inoculum size of less than 3 x 10(4) cells/cm2 . Addition of a crude bovine pituitary extract in MCDB 153 had the greatest impact on hair cell growth . Functional integrity of the cultured hair cells were maintained, since the hair-specific cytoskeletal proteins were expressed in these cells under the present experimental conditions . These desirable conditions could allow selective growth of the hair cells and provide the model system which could be used for the study of hair growth.

Immunol Cell Biol, 1989 Oct, 67 ( Pt 5), 291 - 6
Inhibition of lymphocyte circulation in mice by pertussis toxin; Sewell WA et al.; Pertussis toxin (PT), a protein toxin of Bordetella pertussis, also called pertussigen, has a wide range of biological activities, including the induction of lymphocytosis . This phenomenon was investigated by studying lymphocyte circulation in mice . Lymph node cell suspensions were exposed to PT in vitro and then injected intravenously . A double radiolabel technique was employed, in which PT-treated and control cells were injected into the same animals . The protocol used in these experiments was chosen to demonstrate a direct effect of PT on the injected cells . After exposure to PT in vitro, cells were profoundly excluded from lymph nodes over the succeeding six days . Entry into both mesenteric and peripheral lymph nodes, but not into the spleen was inhibited by PT, and there was an accumulation of the PT-treated cells in the blood . Cells were excluded from the lymph nodes after treatment with as little as 2 ng/mL of PT . This dose was over two orders of magnitude lower than the threshold dose of the same PT preparation required to induce lymphocyte mitogenesis in vitro . The findings in the present communication are consistent with studies using genetically modified PT in which the ADP-ribosylating capacity of the A-subunit was necessary for the effect of PT on lymphocytosis.

Cell Biochem Funct, 1989 Oct, 7(4), 275 - 82
Cytochrome P450 in highly purified suspension of nonparenchymal liver cells; Rich K et al.; Rat liver nonparenchymal cells (NPC) were prepared by pronase digestion and purified on discontinuous gradients on Nycodenz . Morphological and biochemical characterization of cell suspensions showed that they were free of contamination by hepatocytes . We have confirmed the usefulness of pyruvate kinase activity in monitoring the degree of hepatocyte contamination of NPC and we have derived an equation which allows this carry-over to be calculated . Using highly purified suspensions of NPC we have shown that they contain glucose-6-phosphatase in low but detectable levels . Spectrophotometric studies showed that they contain cytochrome P450, with a specific content of 24 +/- 5 pmole mg-1 cell protein . A potential source of error in previous studies was recognized; namely that peroxidase, present in NPC in high concentration, is able to mask the absorption due to cytochrome P450 . Both the presence and inducibility of this enzyme in NPC prepared from rats pretreated with phenobarbital or 3-methylcholanthrene have been confirmed using Western blot analysis.

Hum Pathol, 1989 Oct, 20(10), 987 - 93
Dipeptidylaminopeptidase IV activity in T lymphocyte subsets in B cell non-Hodgkin's lymphomas; Cozzi M et al.; The total T cell population and T cell subsets in ten lymph nodes with reactive lymphoid hyperplasia (RLH) and 23 specimens (21 lymph nodes, one stomach, and one small bowel) involved by histologically and immunohistologically diagnosed B cell non-Hodgkin's lymphomas (NHLs) were determined by reactivity with monoclonal antibodies Leu 4-CD3, Leu 3-CD4, and Leu 2-CD8 in cytospin preparations from cell suspensions . T cell populations were also investigated for the coexpression of dipeptidylaminopeptidase IV (DAP IV) activity, which was visualized simultaneously with cell surface immunostaining by a combined cytochemical and immunocytochemical method . The mean absolute percentage of Leu 4-CD3+ (total T) and Leu 3-CD4+ cell populations was significantly lower in B cell NHL cases than in RLH cases (35% v 54%, P less than .001; 29.5% v 44.4%, P less than .01) . No difference in the mean absolute percentage of the Leu 2-CD8+ T cell subset was found between the RLH cases and the B cell NHL cases classified as other than category A as described by the Working Formulation (WF) of NHLs . The relative percentage of Leu 4-CD3+ and Leu 2-CD8+ cells coexpressing DAP IV reactivity was lower in B cell NHL cases than in RLH cases (27.3% v 39.5%, P less than .05; 13.5% v 24.4%, P less than .10) . There was no difference in the proportion of Leu 3-CD4+ cells expressing DAP IV reactivity between the NHL and RLH groups (34.5% v 36.1%) . Since the mean relative percentage of Leu 2-CD8+ cells expressing DAP IV reactivity in the B cell NHL group in the other than category A according to the WF was lower than that of the RLH group (12.5% v 24.4%), and whereas the mean absolute percentage of total Leu 2-CD8+ cells was similar in the two groups (16.6% and 16.6%), a possible defective role of this Leu 2-CD8+ DAP IV+ subset, at least in B cell NHLs in the other than category A according to the WF, may be hypothesized.

Anal Biochem, 1989 Oct, 182(1), 182 - 6
A dot assay for the erythropoietin receptor using human recombinant 125I-erythropoietin; Vannucchi AM et al.; A dot assay was developed for the detection of membrane receptor(s) for erythropoietin (Ep) . A relatively homogeneous population of cells bearing the receptor for Ep was generated in the spleen of mice made anemic with phenylhydrazine and crude membrane extracts were prepared from spleen cell suspensions . Aliquots of the membrane extracts were applied to microdishes of nitrocellulose in a volume of 4 microliters . After free reactive sites were blocked, the microdishes were incubated for 2 h at 37 degrees C with 125I-labeled human recombinant Ep (125I-rEp), and nitrocellulose bound radioactivity was determined thereafter . Reproducible curves were obtained, and a significant correlation between bound radioactivity and the amount of membrane proteins applied to the nitrocellulose dishes was found . Specific binding was saturable, reaching a plateau at 2.5 nM . Binding parameters of nitrocellulose-immobilized receptor were not significantly different from the values calculated using intact cells . No appreciable binding of 125I-rEp to control membranes at low Ep-receptor content was observed . Among a panel of growth factors, only unlabeled rEp was able to compete for the binding of 125I-rEp to nitrocellulose-immobilized membrane proteins in a dose-dependent fashion . The technique described herein may be of use in the study of the Ep receptor and as an assay for its purification . Moreover, it may also be of general application in the study of receptor-ligand interactions.

Rofo, 1989 Oct, 151(4), 443 - 8
{Computer-assisted ultrasonic B-scan texture analysis of experimental liver tumors}; Layer G et al.; Primary liver cancer has been induced experimentally in female Wistar rats by diaminoazobenzene intoxication or subcapsular liver implantation of Novikoff cell suspensions . The ultrasonic B-scan image of these malignant tumours are described via statistical texture parameters in respect of image brightness, microtexture and macrotexture . A discrimination of the tumour texture profiles is possible with an accuracy of about 80% . There are correlations between ultrasonic image parameters and histomorphological tissue components but there is no specific link between the histological tumour-type and these B-scan data.

Immunobiology, 1989 Oct, 179(4-5), 314 - 27
Isolation and characterization of brain macrophages from the central nervous system of newborn and adult rats and of rats with experimental allergic encephalomyelitis; de Groot CJ et al.; A method has been developed to isolate brain macrophages (M phi) from normal neonatal and adult rats brain cell suspensions, as well as from brain cell suspensions of rat with experimental allergic encephalomyelitis (EAE), by making use of the ability of M phi to adhere to plastic surfaces . The isolated adherent cells were immuno- and enzyme-cytochemically identified . Phagocytic activity and the presence of Fc-IgG receptors were also examined . Approximately 30%-40% of the isolated adherent cells from neonatal rat brain are phagocytic and can be stained with macrophage-specific monoclonal antibodies, suggesting that these cells belong to the monocyte/macrophage lineage . From normal adult rat brain, only a small number of brain M phi could be isolated . A highly purified population of brain M phi was obtained from EAE rat brain . The isolated brain M phi are phagocytic, possess Fc-IgG receptors and rat M phi-associated antigens . Besides these features, the isolated brain M phi also express MHC class II antigens (Ia-antigens), which suggests that M phi may be involved in the regulation of immunological disorders of the CNS . The method reported here for rapidly isolating a large number of blood-monocyte-derived brain M phi from neonatal and adult brain allows an investigation of the precise role of M phi in inflammatory diseases of the central nervous system.

Physiologie, 1989 Oct-Dec, 26(4), 285 - 96
Dynamics and compartmentation of water in certain biosystems; Katona E et al.; Investigation performed by nuclear magnetic resonance and by deuteration on the dynamics of water molecules in mammalian blood cells and in amphibian oocytes, muscles and nerves allowed to reveal several populations of cell water molecules . Following up relaxation and exchange processes in cell suspensions, packed cell pellets and in isolated tissues, characteristics of water dynamics and compartmentation in various biosystems are compared.

Int J Cancer, 1989 Sep 15, 44(3), 494 - 500
Intraperitoneal and subcutaneous xenografts of human ovarian carcinoma in nude mice and their potential in experimental therapy; Massazza G et al.; Human ovarian carcinomas (HOC) were established s.c . and i.p . in nude mice and the biological characteristics were investigated for 4 xenografts . HOC8 and HOC18, derived respectively from a primary tumor of the ovary and a pleural effusion (from 2 different patients) were established s.c . in nude mice . HOC10 and HOC22, derived from the ascites of 2 patients, were directly established as ascites after i.p . injection in nude mice . The s.c . and i.p . growth behavior of the 4 HOC lines was investigated . HOC18, HOC8 and HOC22 cells produced progressively growing tumor after s.c . injection but HOC10 ascites would not grow s.c . The cell suspension derived from HOC18 only produced carcinomatosis upon i.p . injection, while HOC8 cells produced both ascites and carcinomatosis . The 2 ascites HOC10 and HOC22 produced ascites in nude mice, but only HOC22 formed i.p . carcinomatosis . Histopathological characteristics of the patients' primary tumors persisted in nude mice, regardless of the site of tumor implantation . DNA histograms of the xenografts closely matched the patients' tumors and remained stable at different passages . Cisplatin, adriamycin and cyclophosphamide given i.v . were tested against HOC8 and HOC18 growing s.c . and HOC22 and HOC10 growing i.p . HOC8 showed a significant response to DDP and almost no sensitivity to ADR and CTX . HOC18 showed only moderate growth delay with all 3 drugs . Mice bearing HOC10 and HOC22 ascites had a prolonged survival time after DDP and ADR treatment.

Biochim Biophys Acta, 1989 Sep 4, 984(2), 183 - 7
Senescence-induced alteration in cell surface carbohydrates correlated using proton NMR spectroscopy and a lectin-based affinity-binding assay; Busse SC et al.; Changes in the cell surface oligosaccharides in human fetal lung fibroblasts (IMR-90) are studied as the cells progress to senescence using nuclear magnetic resonance spectroscopy (NMR) and a biochemical assay . A lectin-based affinity-binding technique is used which measures the organization of carbohydrates on the cell surface . Proton NMR studies of the water in samples of frozen cell suspensions of young and old cells provide information on the local dynamics of the cell surface by monitoring the motion of bound water . Changes in the lectin binding density and affinity class distribution correlate with a decrease in the water proton linewidth in frozen cells . These observations reflect alterations in the conformation or structure of the cell surface oligosaccharides and local constituent water.

Tohoku J Exp Med, 1989 Sep, 159(1), 23 - 35
Intracellular compartmentalization of fura-2 dye demonstrated by laser-excitation fluorescence microscopy: a problem in measuring cytosolic free calcium concentration using fura-2 fluorescence in vascular smooth muscle cells; Takeuchi K et al.; Recently, we have developed a novel laser-excitation fluorescence microscope system to study intracellular calcium (Ca2+) in individual cultured vascular smooth muscle (VSM) cells using fluorescent indicators for (Ca2+) . In the course of our study, it was shown that the subcellular fluorescence distribution of fura-2 was not homogeneous in VSM cells incubated with the acetoxy-methyl ester form of fura-2, fura-2/AM . The fluorescence appeared spotty or filamentous and resembled in shapes the intracellular organelles, suggesting that there was fura-2 dye compartmentalization in the organelles . To clarify the nature of the subcellular fluorescence, the soluble fraction of cells loaded with the dye was analyzed through high performance liquid chromatography (HPLC) . We also examined the excitation spectra of fluorescence in the soluble fraction, which was compared with that in the cell suspension . Using HPLC, it has been shown that no other than fura-2 was found in the soluble fraction, whereas analyses of excitation spectra have indicated that the membrane fraction contained fura-2/AM or its lipophilic metabolite . On the other hand, indo-1 dye fluorescence showed a diffuse intracellular distribution, but the nuclear region had higher or sometimes lower fluorescence levels than the cytoplasm . The present results suggest that it may be necessary to assess subcellular fura-2 compartmentalization and possible interference by fura-2 AM or its lipophilic metabolite for the accurate measurement of intracellular Ca2+ concentration in VSM cells . It is also suggested that indo-1 may be more suitable for estimating Ca2+ concentration than fura-2 in individual VSM cells.

Neurobiol Aging, 1989 Sep-Oct, 10(5), 640 - 1; discussion 648-50
The use of hollow polymer fibers for the delivery of bioactive molecules to the brain; Springer JE; Experimental studies have provided evidence that bioactive molecules can be delivered to the central nervous system through the use of hollow polymer fibers . These implantable fibers can be used for the delivery of peptide solutions or serve as a carrier device for encapsulating tissue preparations and cell suspensions . This commentary will address the use of these polymer fibers as a potential therapeutic strategy for treating neurodegenerative disorders such as Alzheimer's disease.

J Mol Cell Cardiol, 1989 Sep, 21(9), 877 - 87
Functional impairment in isolated rat hearts induced by activated leukocytes: protective effect of oxygen free radical scavengers; Semb AG et al.; Ischemia-reperfusion activates polymorphonuclear leukocytes (PMN) . Depletion of PMN has been shown to reduce the size of experimental myocardial infarction . We have studied whether PMN activated by phorbol myristate acetate (PMA) would depress function of the isolated rat heart, and if this effect was mediated by oxygen free radicals (OFR) . Cells and/or drugs were added to the perfusate into the aortic cannula for 10 min, followed by a 30 min recovery period . Oxygen free radicals formation was verified by chemiluminescence (CL) . PMA-activated PMN (n = 13) caused CL response of 27,493 +/- 5113 counts (mean +/- S.E.M.) and reduced left ventricular developed pressure (LVDP) to 30 +/- 9% and coronary flow (CF) to 49 +/- 7% of the baseline value at the end of the observation period . Addition of super-oxide dismutase (SOD) and catalase (CAT) (n = 11) reduced the CL response to 5623 +/- 806 counts, but did not influence either LVDP (36 +/- 15%) or CF (51 +/- 18%) . Addition of thiourea (TU) to the activated cell suspension (n = 8) further reduced the CL response (3663 +/- 474 counts), and LVDP was 86 +/- 5% and CF was 87 +/- 3% . When TU + SOD + CAT was mixed with PMN + PMA (n = 11), the CL was almost abolished (117 +/- 21 counts) and LVDP was 73 +/- 8% and CF was 94 +/- 6% . When CF was reduced (n = 7) alike the CF reduction in the hearts receiving PMA + PMA, LVDP was not significantly changed at the end of the observation period (75 +/- 6%) . Unactivated PMN (n = 8) caused minor response of LVDP and CF, similar to PMN + PMA + TU and PMN + PMA + SOD + CAT + TU . PMA alone (n = 8) was cardiotoxic and caused changes similar to PMN + PMA . This effect was not inhibited by scavengers (n = 6) . The supernatant of the PMN + PMA suspension (n = 7) did not impair cardiac function, suggesting that no free PMA was available after mixing with PMN . We conclude that activated PMN in the coronary circulation depressed cardiac function and increased vascular resistance due to OFR production.

Phys Med Biol, 1989 Sep, 34(9), 1153 - 67
Ultrasonic propagation in mammalian cell suspensions based on a shell model; Anson LW et al.; The interaction of ultrasonic waves with individual cells has been modelled on the basis that the cells can be represented by viscous liquid spheres surrounded by a viscoelastic shell (the membrane) immersed in a viscous fluid . The computational model includes thermal waves and requires 22 input parameters . Many of the parameters are not available in the literature and a detailed discussion is given on the procedures by which the values used in the model calculations were chosen . In spite of the arbitrariness of the choice of many of the parameter values, the computations show surprisingly good agreement with experimental measurements of ultrasonic attenuation in animal cell suspensions . The model has been used here to investigate different aspects of the interaction of ultrasound with the cells . It is found that the membrane is important only between 0.5 and 30 MHz and contributes less than 15% to the attenuation . Absorption is shown to be an important feature to include, while the scattering contribution to the attenuation is less than 1% at 3 MHz . The thermal effects are important at frequencies below 1 MHz and contribute some 65% to the attenuation at 100 MHz.

J Biomed Eng, 1989 Sep, 11(5), 375 - 80
Dielectrophoretic study of human erythrocytes; Gopala Krishna G et al.; This paper is concerned with the dielectrophoretic study of human erythrocytes under cylindrical field geometry . The influence of physical variables such as the frequency and voltage of the applied electric field, conductivity of the medium in which the cells are suspended, cell concentration and exposure time of the cell to the non-uniform electric field on dielectrophoretic collection rate (DCR) is determined in a systematic manner . It is interesting to note from the DCR spectrum of human erythrocytes that the DCR is minimum at one frequency, maximum at another and there is practically no yield over a certain frequency range . This may be attributed to the variation of complex dielectric constant of the particle and medium over that frequency range . From the DCR spectrum of different groups, it is clear that DCR behaviour is different in the frequency range from 0.3-1.5 MHz, under similar conditions of temperature, conductivity and concentration of erythrocyte suspension and strength of applied AC field . The response of DCR with voltage of the applied field, concentration of cell suspension and square root of elapsed time of the cells confirms the theory of dielectrophoresis.

J Immunol Methods, 1989 Sep 1, 122(2), 243 - 52
A rapid mechanical vibration procedure for purifying mature (Ia positive) columnar epithelial cells from the mouse small intestine; Kaiserlian D et al.; A mechanical vibration method previously described for isolating gut epithelium was adapted to purify murine small intestinal epithelial cells in single cell suspension with high viability, good yield, optimal purity (99% epithelial cells with no other contaminating intestinal cells), and with preservation of phenotypical and physiological properties of epithelial cells . This rapid and easy procedure did not involve the use of any chelating agent or proteolytic enzyme . Extracted gut epithelial cells were suitable for in vitro functional assays within 45 min . Cell viability, as assessed by both the trypan blue due exclusion test and by titration of lactate dehydrogenase activity in the supernatant was greater than 75% up to 7 h after epithelial cell extraction . Light microscopy analysis of the duodeno-jejunal segment before and after mechanical vibration indicated that villous but not crypt epithelium was obtained using this method . Furthermore 99% of isolated epithelial cells expressed major histocompatibility class II (Ia) antigens indicating that the method was suitable for studies of the antigen presenting capacity of gut enterocytes.

J Leukoc Biol, 1989 Sep, 46(3), 263 - 9
Proliferation of macrophage subpopulations in the adult rat: comparison of various lymphoid organs; Westermann J et al.; Adult male Lewis rats received a single intravenous injection of 5-bromo-2'-deoxyuridine (BRDU) to label all proliferating cells in the S-phase of the cell cycle . Various lymphoid organs were removed 1 and 24 hr after injection to assess local proliferation and migration of newly formed cells, respectively . In cell suspensions, surface staining was performed for macrophage subsets (ED1, ED2, ED3), and the DNA label BRDU was detected by a monoclonal antibody . Local proliferation of ED1+ macrophages occurred in all organs investigated with the exception of the blood . Bone marrow outweighed the other organs by far; in addition to the proliferating ED1+ promonocytes, the bone marrow also contained BRDU-labeled ED2+ macrophages . Newly formed ED1+ monocytes migrated into lymphoid organs such as the mesenteric lymph nodes and spleen where they comprised about 90% of newly formed macrophages . In the spleen, ED3+ macrophages seemed to be renewed by local proliferation, whereas in the mesenteric lymph nodes these cells were replaced by immigration . The heterogeneity of macrophages was further demonstrated by the different renewal of splenic macrophages . ED1+ and ED3+ cells were replaced in a matter of days, whereas it would probably take several months to renew ED2+ cells.

J Cell Physiol, 1989 Sep, 140(3), 491 - 7
Comparison of uptake kinetics in freshly isolated suspensions and short-term primary cultures of rat hepatocytes; Kukongviriyapan V et al.; The apparent kinetics of uptake of various model substrates were examined for hepatocytes in suspension and primary culture up to 72 h . The ability of hepatocytes to take up taurocholate and ouabain was decreased in culture . Vmax for uptake of both substrates diminished rapidly with increasing time in culture . An increase in Km was observed in cultures 6 h after plating, but there was no further change with prolongation of culture time . The decrease of uptake of taurocholate and ouabain during culture may be due to the reduction in the number of transport carriers plus a decrease of affinity of the carrier to substrates . The nonsaturable component of cadmium uptake was much reduced in cultured cells compared with the suspensions . The saturable process was lower in 6 h culture but increased to a level comparable with the fresh cells at longer culture time . No significant change was found in the Km between suspensions and cultures . Uptake of alpha-aminoisobutyric acid was greater in culture while that of 3-O-methyl-D-glucose was relatively stable but about one-half that found in cell suspension . Thus, uptake of two substrates, taurocholate and ouabain, is clearly compromised with increasing time in primary culture, while uptake of the other substrates does not reflect such a dramatic decrease . It is therefore apparent that the cell preparation of choice in uptake studies depends on the substrate and the nature of the experiments.

Am J Kidney Dis, 1989 Sep, 14(3), 200 - 3
Lead increases red cell sodium-lithium countertransport; Batuman V et al.; The effect of 1, 3, 5, and 20 mumol/L lead on normal red cell sodium-lithium countertransport was studied in vitro . Red cell suspensions incubated with lead had increased sodium-lithium countertransport at all concentration levels compared with paired, unleaded controls when all groups were evaluated by analysis of covariance (F = 19.2, P less than 0.001) . The effect of lead was concentration dependent (r = 0.998, P less than 0.001) . These observations suggest that abnormalities in sodium transport are involved in the pathogenesis of lead-induced hypertension . Because increased red cell sodium-lithium countertransport is characteristic of essential hypertension, these observations further suggest that lead-induced and essential hypertension may share common pathophysiological mechanisms.

Int J Hyperthermia, 1989 Sep-Oct, 5(5), 617 - 24
The effect of preirradiated tumour bed on the response of a murine fibrosarcoma to elevated temperatures; Urano M et al.; The effect of pretransplantation irradiation of tumour beds (tumour bed effect or TBE) on the response of tumours to elevated temperatures and on the kinetics of thermo-tolerance was studied . Animal tumours were early generation isotransplants of a fibro-sarcoma, FSa-II in C3Hf/Sed mice . The tumour bed or murine foot was irradiated with 0.8 or 16 Gy in air, and tumour cell suspensions were transplanted 1-35 days thereafter . Hyperthermia of various durations was given in a 45.5 degrees C water bath when tumours reached an average diameter of 6 mm (110 mm3), and the tumour growth (TG) time to reach 500 mm3 was obtained . Dose-response curves between the TG time and the treatment time were less steep for tumours in preirradiated tumour beds than for tumours in non-irradiated tumour beds, indicating that the tumours in preirradiated tumour beds were more resistant to hyperthermia compared to the tumours in normal tumour beds . This resistance appeared to increase with increasing preirradiation dose . Thermotolerance was equally developed in tumours in normal and preirradiated tumour beds . The time required to develop the maximum thermotolerance was identical for both tumours . This indicated that the TBE has no effect on the kinetics of thermotolerance . The implication of these results in clinical trials may be that the tumour which recurred after radiotherapy may not be a good candidate for hyperthermia.

Exp Cell Res, 1989 Sep, 184(1), 72 - 80
Homologous but not heterologous contact increases the insulin secretion of individual pancreatic B-cells; Bosco D et al.; To assess whether and how specifically contact influences the functioning of differentiated cells, we have studied the secretion of adult pancreatic B-cells as a function of aggregation to either homologous B-cells or other heterologous endocrine islet cell types, all present in a mixed cell suspension . Using an immunological plaque assay for insulin, we have quantitated the proportion of single and aggregated B-cells inducing the formation of a hemolytic plaque (a reflection of the size of the secreting cell population) and the area of these plaques (a reflection of the hormonal output of individual cells or aggregates) after a 30-min stimulation by 16.7 mM glucose . By taking into account the number of B-cells within the aggregates, we have calculated from these data the insulin output on a per B-cell basis . We show here that the homologous contact between companion B-cells promotes the recruitment of secreting B-cells and increases their individual secretion of insulin twofold over that of single B-cells . By contrast, heterologous B- to non-B-cell contact was not effective in enhancing the recruitment of secreting B-cells and in promoting their individual secretion . These findings show that a highly differentiated cell function, such as insulin secretion, is controlled specifically by homologous cell to cell contacts.

Nippon Hifuka Gakkai Zasshi, 1989 Sep, 99(10), 1067 - 73
{The functional maturation of Langerhans cells in CTL induction during tissue culture}; Daita Y et al.; In the previous report, we investigated the effects of tissue culture on the APC function of murine epidermal Langerhans cells (LC) in the induction of allo-CTL in vitro, and found that (1) cultured ear LC expressed increased amounts of Ia antigens on their cell surface, and (2) they induced extremely enhanced levels of CTL over those produced by freshly prepared ear LC and also (3) cultured tail LC were proved to be able to induce CTL for the first time . It seemed that tissue culture decreased the functional heterogeneity among the murine ear LC and tail LC in addition to that among Ia+ APC in spleen and in epidermis . In this study, we investigated the culture conditions that increase the APC function of LC . Our data indicate that LC cultured with dermal components exhibited more enhanced APC function than LC cultured in single cell suspension with only epidermal cells . Recent studies indicate that IL-1 and GM-CSF, which keratinocytes release, are essential for freshly prepared LC to mature into highly efficient APC that resemble splenic dendritic cells . We found dermal factors are more important than epidermal ones for LC to mature in tissue culture.

Vet Pathol, 1989 Sep, 26(5), 396 - 408
Investigation of bovine gut associated lymphoid tissue (GALT) using monoclonal antibodies against bovine lymphocytes; Parsons KR et al.; Gut associated lymphoid tissue of the small and large intestine of calves and cows has been compared morphologically and quantitatively using monoclonal antibodies to bovine lymphocytes . B cells were significantly decreased in the ileum of the cow compared to the calf . Significantly increased numbers of T cells were present in cell suspensions of all lymphoid areas of the cow compared to the calf . T lymphocyte subsets were quantified into cryostat sections of lymphoid tissues expressing BoT4, and BoT8 antigens demonstrated increased numbers in follicular and dome areas of the discrete Peyer's patches of the small and large intestine of the cow . BoT4+, BoT8+, and the non-BoT4/BoT8+ T cell subsets were increased in the mucosa of the cow as compared to the calf . Similarities in structure and lymphocyte composition of the discrete Peyer's patches of the small intestine, cecum and colon and isolated single follicles in the large intestine suggest similar functional properties.

Drug Metab Dispos, 1989 Sep-Oct, 17(5), 481 - 6
Metabolism of acrylonitrile to 2-cyanoethylene oxide in F-344 rat liver microsomes, lung microsomes, and lung cells; Roberts AE et al.; The metabolism of acrylonitrile to the epoxide, 2-cyanoethylene oxide (ANO) was examined in rat liver microsomes, lung microsomes, and isolated enriched lung cell preparations . GC/high resolution MS was used to quantitate ANO in microsomal and cellular extracts by monitoring the fragment ion C2H3N (m/z 41.0265) . The limit of detection was 0.05 pmol of ANO/0.5 microliter of standard solution, microsomal extract, or cellular extract injected onto the column, and the linear range of analysis was 0.05 to 12.5 pmol of ANO . Kinetic parameters of Vmax, V/K, and Km were calculated for microsomal ANO formation . Liver microsomes were quantitatively more active than lung microsomes on a mg of protein basis . The Vmax (pmol of ANO formed/min/mg of protein) was 666.61 for liver and 45.07 for lung microsomes . The V/K (pmol of ANO/min/mg of protein/microM) was 12.83 for liver and 0.02 for lung microsomes . The apparent Km was 51.93 microM and 1853.83 microM for liver and lung microsomes, respectively . When calculated as nmol of ANO formed/min/nmol of microsomal P-450, the Vmax for lung was equivalent to the Vmax for liver . ANO formation in the rat lung was cell specific . The rates of metabolism in the Clara cell-enriched fraction, the alveolar type II cell-enriched fraction, and the cell suspension were 2.55, 0.38, and 0.67 pmol of ANO formed/min/mg of protein, respectively . No metabolism was observed in the endothelial (small) cell-enriched fraction or in the alveolar macrophages . The results suggest that the lung contributes to the metabolism and disposition of inhaled acrylonitrile.

Exp Neurol, 1989 Sep, 105(3), 251 - 9
Dopamine released from mesencephalic transplants restores modulation of striatal acetylcholine release after neonatal 6-hydroxydopamine: an in vitro analysis; Carder RK et al.; After chemical lesions which destroy the nigrostriatal dopamine pathway, transplants rich in dopamine neurons innervate the striatum and, with appropriate stimulation, drive host motor behaviors normally mediated by dopamine . We wished to determine whether dopamine released from the transplant also reinstated dopaminergic inhibition of striatal acetylcholine release . Three-day-old rat pups received bilateral intraventricular injections of 6-hydroxydopamine . Three days later cell suspensions prepared form embryonic ventral mesencephalon were injected unilaterally into the striatum . Tail pinch and amphetamine were able to elicit contralateral turning in many of these animals . Only those animals which rotated greater than or equal to 5 turns/min were included for further analysis . Subsequent assays indicated that 6-hydroxydopamine had depleted striatal dopamine to 4% of control and that the transplant had increased dopamine levels to 11% of control . Superfused striatal slices were stimulated (8 Hz, 1 min) and then exposed to amphetamine (10 microM, 3 min) . The slice released dopamine, as measured by HPLC, and acetylcholine, as measured by tritium efflux after preincubation with {3H}choline . Moreover, the release of acetylcholine was inhibited by endogenous dopamine as indicated by the ability of sulpiride (1 microM) to increase tritium efflux . Striatal slices prepared from lesioned animals showed a reduction in dopamine overflow in response to both electrical stimulation (0.6% of control) and amphetamine (1% of control), and a decrease in the ability of sulpiride to increase electrically evoked acetylcholine overflow (12% of control) . Transplantation partially restored the dopaminergic response to electrical stimulation (21% of control), and amphetamine (15% of control) and fully restored the sulpiride-induced increase in acetylcholine overflow (98% of control).(ABSTRACT TRUNCATED AT 250 WORDS)

Development, 1989 Sep, 107(1), 153 - 63
Biochemical differentiation in a mutant of Dictyostelium discoideum defective in cyclic AMP chemotaxis and in intercellular cohesion; Srinivas UK et al.; A temperature-sensitive mutant of Dictyostelium discoideum has been isolated based on its lack of chemotaxis toward cyclic AMP at the restrictive temperature, 27 degrees C . The mutant develops normally at the permissive temperature, 22 degrees C, but fails to aggregate or complete development at the restrictive temperature . The temperature-sensitive phenotype can be bypassed by allowing cultures to grown into late log phase or to starve for 60-90 min at 22 degrees C prior to a shift to 27 degrees C . At 27 degrees C, the mutant overproduces cell surface cyclic AMP receptors of both high and low affinity and is capable of spontaneous oscillations in light scattering in cell suspensions . Despite its complete lack of morphological development, the mutant undergoes extensive biochemical differentiation . At the onset of starvation, it shows increased levels of N-acetylglucosaminidase, it express cyclic AMP receptors at the normal time and, although somewhat slowly, suppresses those receptors as if aggregation had been achieved . Metabolic pulse labellings with {35S}methionine revealed that the mutant at 27 degrees C displays the same changes in the patterns of newly synthesized proteins observed during the vegetative-to-aggregation and the aggregation-to-slug stages of normal development . The only clear difference from wild type was the failure of the culmination-stage isozyme of beta-glucosidase to appear . The mutant is defective in establishment of intercellular cohesion mechanisms, correlated with poor agglutination by concanavalin A, at the restrictive temperature . The properties of the mutant place severe constraints on models regarding the role of chemoreception and intercellular cohesion in regulation of gene expression.

Kokyu To Junkan, 1989 Sep, 37(9), 997 - 1002
{New quantitative method for non-invasive monitoring of tissue blood oxygenation by near infrared spectrophotometry}; Tamura M et al.; The near infrared absorption spectra was measured with the transmitted light through rat brain under the various condition . The absorbance changes below 780 nm were attributable to hemoglobin (Hb) in the brain tissue, whereas those above 780 nm were associated with both Hb and cytochrome oxidase . To eliminate possible interference from cyt . oxidase, two wavelengths, 750 nm and 780 nm, were used to measure Hb oxygenation in the tissue . The absorbance changes in human blood cell suspensions were measured with changes in hematocrit values in the optical cuvette . At two wavelengths 750 nm and 780 nm, there was a linear relationship between absorbance changes and hematocrit values . Through these in vitro studies, the following equation (1) and (2) were obtained to monitor quantitatively the changes of oxy-Hb content (delta Hb O2) and total-Hb content (delta Hb Vol.) in the living tissue . These are (1) delta Hb O2 = -1.15 delta A 750 + 1.39 delta A 780, (2) delta Hb Vol . = -0.29 delta A 750 + 0.59 delta A 780 . The studies using these equations showed that the oxy-Hb content in the brain was decreased as the O2 concentration in inspired gas was lowered with a half of Hb deoxygenated at 7% O2 . The reliability of these equations was examined under the various conditions in situ such as CO2 inhalation, intravenous injection of Ca2+-blocker nicardipine, hemorrhage and retransfusion . These results confirmed that these equations derived from in vitro studies, were successfully applied to the in situ measurements of the oxygenation state of Hb in the living tissues.

J Cell Physiol, 1989 Sep, 140(3), 565 - 76
Serum-free culture of normal human melanocytes: growth kinetics and growth factor requirements; Pittelkow MR et al.; Normal human epidermal melanocytes were selectively propagated from mixed (keratinocyte-melanocyte) cultures and primary epidermal cell suspensions in serum-free medium, MCDB 153 containing insulin, bovine pituitary extract (BPE), phorbol-12-myristate-13-acetate (PMA), ethanolamine, phosphoethanolamine, and hydrocortisone . Neonatal foreskin melanocytes (NFMs) replicated more readily than adult melanocytes in culture . Early passage NFMs grown in serum-free medium exhibited a population generation time of 24-48 hours . NFMs assumed a less dendritic appearance and were less pigmented than adult melanocytes . PMA or other protein kinase C-activating phorbol esters significantly enhanced mitogenesis of NFMs; however, cAMP-elevating agents were not required for efficient replication of NFMs . Basic fibroblast growth factor (bFGF) was a potent mitogen for NFMs and replaced the requirement for BPE in the culture medium . NFMs expressed a single class of specific, high-affinity receptors for bFGF, exhibiting a Kd = 3 x 10(-11) M and approximately 76,500 receptors/cell . Neither EGF nor TGF-alpha were mitogenic for NFMs, and TGF-beta reversibly inhibited NFM growth . Rapidly growing, early passage NFMs were shown to have cell cycle times of 19.5, 7.5, and 9 hours for G1, S, and G2/M phases of the cell cycle, respectively . Culture of NFMs to confluence or depletion of growth factors from the culture medium caused reversible, G1 phase-specific, cell cycle growth arrest . Senescence of NFMs was associated with irreversible growth arrest in the G1 phase after 40-45 population doublings in culture . Our data demonstrate that basal medium MCDB 153 can be supplemented with defined factors to cultivate selectively two major constituent cell types of the epidermis, the melanocyte and the keratinocyte.

J Cell Physiol, 1989 Sep, 140(3), 505 - 11
Oxygen gradients in CHO cells: measurement and characterization by electron spin resonance; Glockner JF et al.; The concentration of oxygen within cells is important in many physiological and pathological processes, but such oxygen-dependent processes are generally studied as a function of the concentration of extracellular oxygen, due to a lack of suitable methods . Using a newly developed technique based on ESR spectroscopy, we show that respiration stimulation of a cell suspension can result in a significant difference between average intracellular and extracellular concentrations of oxygen . These results indicate that studies of oxygen-dependent phenomena in cells may require measurement of intracellular oxygen concentrations and imply that there are mechanisms in cells that restrict the free diffusion of oxygen.

Eur J Biochem, 1989 Sep 1, 184(1), 223 - 32
The role of sodium ions in methanogenesis . Formaldehyde oxidation to CO2 and 2H2 in methanogenic bacteria is coupled with primary electrogenic Na+ translocation at a stoichiometry of 2-3 Na+/CO2; Kaesler B et al.; Cell suspensions of Methanosarcina barkeri were found to oxidize formaldehyde to CO2 and 2H2 (delta G0' = -27 kJ/mol CO2), when methanogenesis was inhibited by 2-bromoethanesulfonate . We report here that this reaction is coupled with (a) primary electrogenic Na+ translocation at a stoichiometry of 2-3 Na+/CO2, (b) with secondary H+ translocation via a Na+/H+ antiporter and (c) with ATP synthesis driven by an electrochemical proton potential . This is concluded from the following findings . Formaldehyde oxidation to CO2 and 2H2 was dependent on Na+ ions, 2-3 mol Na+/mol formaldehyde oxidized were extruded . Na+ translocation was inhibited by Na+ ionophores, but not affected by protonophores of Na+/H+ antiport inhibitors . Formaldehyde oxidation was associated with the build up of a membrane potential in the order of 100 mV (inside negative), which could be dissipated by sodium ionophores rather than by protonophores . Formaldehyde oxidation was coupled with ATP synthesis, which could be inhibited by Na+ ionophores, Na+/H+ antiport inhibitors, by protonophores and by the H+-translocating-ATP-synthase inhibitor, dicyclohexylcarbodiimide . With cell suspensions of Methanobacterium thermoautotrophicum similar results were obtained.

J Exp Med, 1989 Sep 1, 170(3), 679 - 90
In vivo administration of histoincompatible lymphocytes leads to rapid functional deletion of cytotoxic T lymphocyte precursors; Martin DR et al.; It is well established that a single intravenous injection of F1 lymphocytes can rapidly and specifically reduce the ability of a parental recipient to generate CTL against donor alloantigens in a subsequent MLR . By fluorescently labeling the injected cells, we have been able to identify, and if desired, remove them in cell suspensions prepared from recipient spleen and lymph node . The injected cells, whether F1 or syngeneic, appeared to form part of the normal recirculating pool . Removal of injected F1 cells from responder lymph node or spleen cell suspensions had no effect on the response reduction observed in the 5-d in vitro MLR (typically 80% reduction for responder cells taken 2 d after injection of F1 cells) . When the frequency of CTL precursors (CTLp) was measured by limiting dilution, it was reduced to the same degree as the MLR response, implying that response reduction is due to a reduction in the number of activatable CTL in the responder cell suspension . An equal mixture of responder cells from treated (i.e., F1 injected) and control mice gave a measured CTLp frequency equivalent to the average of the separate frequencies, implying the absence of suppressor cells active in vitro . Labeled F1 cells recovered from a first recipient could be used to induce response reduction in a second recipient . The results are discussed in terms of APCs that functionally delete rather than stimulate CTLp that recognize them (i.e., a "veto mechanism") . These experiments appear to rule out a role for in vivo-induced suppressor cells up to 8 d after injection of semiallogeneic cells but do not address the question of whether they are induced at later times.

J Immunol, 1989 Sep 1, 143(5), 1447 - 52
Tolerance induction of allo-class II H-2 antigen-reactive L3T4+ helper T cells and prolonged survival of the corresponding class II H-2-disparate skin graft; Hori S et al.; The present study investigates the effects of i.v . presensitization with class II H-2-disparate allogeneic cells on various L3T4+ T cell functions including the capability of rejecting the corresponding allogeneic skin graft . C57BL/6 (B6) mice were i.v . presensitized with class II H-2 disparate B6-C-H-2bm12 (bm12) spleen cells . Such presensitization did not affect the bm12-specific L3T4+ T cell-mediated proliferative and interleukin 2 (IL-2)-producing capacities . A single cell suspension of (B6 x bm12)F1 spleen cells was depleted of APC by two round-passages over Sephadex G-10 columns . This APC-depleted fraction of (B6 x bm12)F1 cells failed to stimulate B6 responding cells in mixed lymphocyte reactions (MLR) . The addition of recombinant IL-1 to the MLR restored anti-bm12 MLR responses, indicating that APC-depleted (B6 x bm12)F1 cells bear bm12 alloantigens but are unable to stimulate B6 anti-bm12 L3T4+ T cells . A single i.v . administration of APC-depleted (B6 x bm12)F1 cells into B6 mice resulted in almost complete abrogation of the capacity of recipient B6 lymphoid cells to give anti-bm12 MLR and IL2 production . This suppression was bm12 alloantigen-specific and attributed to the elimination or functional impairment of anti-bm12 T cell clones rather than the induction of suppressor cells . The tolerance was also observed in graft-rejection responses . The strikingly prolonged survival of bm12 skin grafts was produced when grafts were implanted into B6 mice which had been presensitized with APC-depleted, but not with untreated (B6 x bm12)F1 spleen cells . These results indicate that allo-class II H-2 antigen-reactive L3T4+ T cells are rendered tolerant by i.v . presensitization with APC-depleted fraction of the corresponding allogeneic cells.

Am J Respir Cell Mol Biol, 1989 Sep, 1(3), 237 - 44
Arachidonate 12-lipoxygenase and cyclooxygenase: PGE isomerase are predominant pathways for oxygenation in bovine tracheal epithelial cells; Hansbrough JR et al.; The capacity of bovine tracheal epithelial cells to convert arachidonic acid to oxygenation products with potential biologic activity was studied in homogeneous preparations of isolated cells . Purified epithelial cell suspensions were incubated with radiolabeled arachidonic acid, and oxygenated metabolites were identified using high-pressure liquid chromatography and gas chromatography-mass spectrometry . The cells released predominantly two products during incubation with 0.3 to 150 microM arachidonic acid for 1 to 60 min at 37 degrees C: prostaglandin E2 (PGE2) and 12-hydroxyeicosatetraenoic acid (12-HETE) . Concentration-response curves for the two products yielded half-maximal effects at 2 and 45 microM arachidonic acid, respectively . Stereochemical analysis by chiral-phase high-pressure liquid chromatography demonstrated that the epithelial 12-HETE consisted exclusively of the 12(S) isomer, providing supporting evidence that it was derived from an arachidonate 12-lipoxygenase . Epithelial cells prelabeled with arachidonic acid and incubated with 5 microM A23187 to stimulate endogenous arachidonic acid metabolism also released two predominant products with the chromatographic properties of PGE2 and 12-HETE . The findings demonstrate that bovine tracheal epithelial cells express both a cyclooxygenase:PGE isomerase and a 12-lipoxygenase pathway and therefore implicate this pathway as a new source of epithelial cell mediators.

Endocrinology, 1989 Sep, 125(3), 1204 - 10
Parathyroid hormone-related peptide transiently increases cytosolic calcium in osteoblast-like cells: comparison with parathyroid hormone; Civitelli R et al.; PTH-related protein (PTHrP), similarly to PTH, stimulates cAMP production in target tissues . However, different potencies have been observed for the two peptides in some biological assays, suggesting that cAMP-independent second messenger pathways might be involved in PTHrP signal transduction . This hypothesis was tested in the osteogenic sarcoma cell line UMR 106-01 . Addition of PTHrP-(1-34) to cell suspensions loaded with the Ca2+ indicator indo-1 produced a transient dose-dependent increase in intracellular calcium ({Ca2+}i), with a maximal effect at 2 x 10(-7) M and an ED0.5 at about 4 x 10(-8) M . The amplitude and duration of the transients were similar to those induced by equimolar concentrations of bovine PTH-(1-34) (bPTH), and the dose-responses of the two peptides completely overlapped . Both full-length peptides, PTHrP-(1-141) and bPTH-(1-84), produced effects identical to those observed with the 1-34 fragments . Homologous and heterologous desensitization to both PTHrP-(1-34) and PTHrP-(1-141) occurred when the cells were prestimulated with equimolar or 10-fold lower doses of either PTHrP-(1-34) or bPTH-(1-34) . Desensitization to bPTH-(1-34) was also observed when cells were prestimulated with PTHrP-(1-34) . Furthermore, pretreatment with either bPTH-(3-34) or {Nle8,18, Tyr34}bPTH-(3-34) amide did not affect {Ca2+}i, but reduced the response to PTHrP-(1-34) by 55 +/- 10% (n = 3) and 67 +/- 8% (n = 3), respectively . The PTHrP-(1-34)-induced {Ca2+}i transient was not substantially affected by either extracellular Ca2+ chelation by EGTA or pretreatment with diltiazem, and nitrendipine only partially inhibited the {Ca2+}i response to PTHrP-(1-34) by about 10% . These results indicate that in osteoblastic cells PTHrP mobilizes Ca2+ from an intracellular storage pool with potency equal to that of PTH, and that the two hormones interact with the same receptor.

J Clin Immunol, 1989 Sep, 9(5), 421 - 8
Preferential loss of Leu 8-, CD45R, HLA-DR+ CD8 cell subsets during in vitro culture of mononuclear cells from human immunodeficiency virus type I (HIV)-seropositive former blood donors; Prince HE et al.; Recent data from our laboratory showed that the CD4:CD8 cell ratio increased significantly during in vitro culture of unstimulated mononuclear cells (MC) from HIV-infected persons . To test the hypothesis that this increase reflected a decline in CD8 cell levels, changes in CD4 and CD8 cell levels during culture of MC were assessed quantitatively . These analyses were accomplished using a Spectrum III flow cytometer, which analyzes a constant volume (0.02 ml) of cell suspension . The number of cells counted within this volume (termed the sip count) thus reflects the cell number in the suspension . To establish day 0 sip counts, aliquots consisting of 200,000 lymphocytes were treated with monoclonal antibodies, resuspended in 1 ml of buffer, and analyzed . Also on day 0, identical cell aliquots were placed in microtiter wells and cultured for 3 days . Cells retrieved from individual wells were then analyzed as on day 0 . The mean relative recoveries (RR) of lymphocytes, CD4 cells, and CD8 cells were significantly lower in the HIV group (N = 28) than in the control group (N = 26) . For the HIV group, CD8 cell RR was significantly lower than CD4 cell RR . Dual-color analyses showed that CD4 cell loss in the HIV group did not occur preferentially within CD4 subsets defined by Leu 8 or CD45R expression . Similarly, CD8 cell loss did not occur preferentially within CD8 subsets defined by Leu 7 expression . In contrast, CD8 cell loss did preferentially affect Leu 8-, CD45R-, and HLA-DR+ CD8 subsets, compared to the reciprocal CD8 subsets.(ABSTRACT TRUNCATED AT 250 WORDS)

Nippon Geka Gakkai Zasshi, 1989 Sep, 90(9), 1451 - 4
{Adoptive immunotherapy of advanced cancer patients with immune RNA-sensitized lymphocytes}; Fukushima M et al.; Tumor cell suspension was prepared from resected tumor tissue of various cancer patients . I-RNA was extracted from lymphoid tissues of rabbits immunized with each tumor cell and CFA . Autologous (in some cases, allogeneic) lymphocytes were prepared with blood cell separator and incubated with I-RNA, then, returned to himself . Twenty five cases of non-curatively resected gastric cancer (group A), 27 cases of stage II-IV of esophageal carcinoma (group B), 21 of lung cancer (group C), 14 of colorectal cancer (group D) and 37 cases with metastatic lesions (group E) were treated with this schedule . Five year cumulative survival rate was 21% in group A, 23% in group B, 46% in group C and 61% in group D, respectively . One case of CR and 5 of PR were recognized in group E . In many cases of the responder, lymphocyte count, ratio of Leu 4+, 3a+ subset in the peripheral bloods increased and skin reaction to autologous tumor cell extract, LAI and LMI became to positive . Interferon activity was also increased in the responder . It was reported on the preliminary study of combination treatment with I-RNA sensitized lymphocytes and IL-2.

Clin Sci (Lond), 1989 Sep, 77(3), 297 - 304
Mast cell heterogeneity in human lung tissue; Van Overveld FJ et al.; 1 . In this study mast cells were found to comprise 2.1% of total cells recovered by enzymatic digestion of human lung tissue . 2 . This mast cell population consisted of 79% formalin-sensitive, Alcian Blue-positive mast cells and 21% formalin-insensitive, Alcian Blue-positive mast cells . 3 . By the use of centrifugal elutriation and subsequent Percoll gradient centrifugation, separate mixed cell populations could be obtained in which the mast cell constituents were either of the formalin-sensitive or -insensitive type . 4 . Cell suspensions in which formalin-sensitive cells comprised 97% of mast cells contained approximately 1.34 pg of histamine per mast cell, whereas in preparations in which mast cells were 84% formalin-resistant the histamine content was approximately 4.17 pg of histamine per mast cell . 5 . The histamine release upon anti-immunoglobulin E challenge of formalin-sensitive mast cells was greater than the release by formalin-insensitive mast cells . 6 . After challenge with opsonized zymosan, only formalin-sensitive mast cells were able to release histamine . 7 . Leukotriene C4 release was observed when formalin-sensitive mast cells were challenged with anti-immunoglobulin E . Formalin-insensitive mast cells showed no release of leukotriene C4 . 8 . Prostaglandin D2 release was observed when formalin-insensitive mast cells were challenged with anti-immunoglobulin E . Formalin-sensitive mast cells showed no release of prostaglandin D2.

J Immunol, 1989 Aug 15, 143(4), 1215 - 22
Effect of IL-7 on the growth of fetal thymocytes in culture; Watson JD et al.; The effects of IL-7 on the in vitro growth and differentiation of day 12 to 14 murine fetal thymocytes were examined in three culture systems . In single cell suspension cultures, IL-7 and IL-2 induced a DNA synthetic response in a short term (1 day) assay, but neither cytokine supported continued cell growth . In conventional fetal thymus organ cultures, the addition of exogenous IL-7 resulted in a twofold increase in cell number over that which normally develops in unsupplemented fetal thymus organ cultures during a 7-day period . The most striking effects of IL-7 were noted in lobe submersion cultures (LSC), a system in which thymocyte growth was totally dependent on the addition of exogenous cytokine . Cells proliferated for a period of approximately 2 wk in IL-7, and cell viability could be maintained even longer . A high percentage of cells recovered after 7 to 14 days from IL-7-supplemented LSC resembled the earliest detectable fetal thymocytes with regard to cell surface markers: they expressed Pgp-1, lacked CD4, CD8, and CD3 and many expressed the IL-2R . These results suggest that IL-7 promotes the growth of cells that occur early in the T cell lineage . Cell populations recovered from LSC supplemented with IL-7 and IL-2 exhibited differential expression of some surface markers, particularly CD3 and NK1.1 . In addition, cells from LSC supplemented with IL-7 were found to proliferate upon subsequent exposure to IL-2, but cells from LSC containing exogenous IL-2 were no longer responsive to IL-7 . These results imply that IL-7 and IL-2 may act at different stages of thymocyte differentiation . Together with previous observations of IL-7-specific mRNA expression in the thymus, this study provides evidence highly suggestive of a pivotal role for IL-7 in T cell development.

Cell Biol Int Rep, 1989 Aug, 13(8), 701 - 10
Isolation of a pure suspension of proximal tubular cells from the rabbit kidney: morphological, biochemical and metabolic assessment; Toutain H et al.; A pure suspension of proximal tubule cells from the rabbit kidney was isolated by a new procedure, without proteolytic enzyme treatment . Electron microscopic examination revealed that these intact cells had long microvilli . All the marker enzymes located in the proximal tubule were higher in the isolated cells compared with renal cortex . Adenylate cyclase activity of the isolated cells was increased by PTH and NaF stimulations, while other hormones had minor effects . This isolated cell suspension showed a high respiration rate, a linear glucose production and a normal cell ATP level . All these results confirmed the isolation of viable proximal tubular cells with high metabolic capacities from the rabbit kidney.

Biomaterials, 1989 Aug, 10(6), 380 - 6
Microencapsulation of human diploid fibroblasts in cationic polyacrylates; Mallabone CL et al.; Human diploid fibroblasts and Chinese hamster ovary cells were encapsulated in several copolymers of dimethylaminoethyl methacrylate with methacrylic acid and/or methyl methacrylate . Copolymers containing 16 to 25% dimethylaminoethyl methacrylate and less than or equal to 2.2% methacrylic acid (based on monomer mol%) supported human diploid fibroblast growth when the polymer was cast as a film on glass or polystyrene . The cells survived encapsulation and grew, but growth was only observed in those capsules which appeared to be flawed; the flaws were detected as an early loss of fluorescence, due to leakage of the FITC-dextran added as a marker to the encapsulated cell suspension . Presumably the capsule wall had too low a permeability to allow for unrestricted growth . Chinese hamster ovary cells behaved similarly in dimethylaminoethyl methacrylate/methyl methacrylate capsules . Increasing the water content, by addition of methacrylic acid, did not improve matters, since these materials were not as good a substrate for cell growth as the others . Preparing materials that are sufficiently permeable, with low toxicity and high processability and which support the growth of anchorage-dependent cells is difficult, yet it remains an appropriate goal for further study.

Clin Exp Immunol, 1989 Aug, 77(2), 168 - 74
T lymphocyte activation in systemic lupus erythematosus analysed by proliferative response to nucleoplasmic proteins on nitrocellulose immunoblots; Pham BN et al.; Polyclonal B cell activity in systemic lupus erythematosus (SLE) may be under T cell control . The use of nitrocellulose immunoblots for the analysis of recognition by peripheral blood lymphocytes of nucleoplasmic proteins in SLE patients led to the characterization of significant proliferative responses to 68K (U1 RNP); SS-B; B-B' and D (Sm) antigen in 15 of 20 patients . Variations of proliferative response were parallel to disease activity over a follow-up period of greater than or equal to 6 months, conferring some prognostic value to the assay of lymphocyte response to nucleoplasmic antigens . The pattern of reactivity differs from the corresponding serum antibody profile, and purified T cell suspensions (greater than 95% pure) were shown to proliferate in response to soluble nucleoplasmic antigens, indicating that T and B cell repertoires against nucleoplasmic proteins may differ . This suggests that activated helper T cells contribute to the fine modulation of B cell reactivity to subcellular particles to determine the particular antibody profile of the patients.

Immunol Cell Biol, 1989 Aug, 67 ( Pt 4), 215 - 21
Ontogeny, morphology and tissue distribution of a unique subset of CD4-CD8- sheep T lymphocytes; McClure SJ et al.; The morphology, distribution and ontogeny of the 197+ T lymphocyte subpopulation is described . Cells were identified by immunofluorescent and immunoenzymic staining of cell suspensions and tissue sections . In the circulation, 197+ cells are small and regular, indistinguishable in size from peripheral CD4+ and CD8+ cells, with little cytoplasm and a large amount of condensed chromatin . In the post-natal animal they are unevenly distributed, being most common in the circulatory pathways, particularly in the blood . In the thymus they chiefly localize in the regions around Hassal's corpuscles and medullary blood vessels . They are totally absent from B cell areas in all tissues . They are the last of the T cells to appear in ontogeny, and are first detected in the thymus . Their frequency in blood is age-dependent with peak levels occurring perinatally . There is a post-natal decline in the frequency of 197+ cells in ileocaecal but not in prescapular lymph nodes . We conclude that these T cells differ from the more commonly described T cells, not only in their surface expression of the CD4, CD8 and T19 antigens, but also in the time of their first appearance, their age-related prevalence and their distribution between and within tissues.

Clin Exp Immunol, 1989 Aug, 77(2), 263 - 8
Isolation and functional studies of granulated lymphocytes in first trimester human decidua; Ritson A et al.; Granulated lymphoid cells (CD2+, CD7+, CD38+, NKH1+, CD3-, CD5-, CD4-, CD8-, CD25-) are prominent in human endometrial stroma in the late secretory phase of the menstrual cycle and in early pregnancy, and may play an important role in implantation and placentation . Cell suspensions enriched for granulated lymphoid cells were prepared from first trimester human decidua using a panning technique; cells were labelled with the monoclonal antibody NKH1 and separated by adherence to immunoglobulin-coated plates . The enriched cells were characterized with a panel of monoclonal antibodies using an indirect immuno-alkaline phosphatase method, and subjected to various functional assays . Most cells in the enriched preparations showed the characteristic morphology of granulated lymphocytes in smears stained with toluidine blue or May Grunwald Giemsa . CD45+ cells were obtained up to 98 +/- 1% purity (n = 10) and CD2+ cells were enriched up to 84 +/- 4% . The enriched populations were efficient effectors in a K562 chromium-release assay but showed minimal proliferative response to phytohaemagglutinin, concanavalin A, ionomycin and phorbol 12, 13 dibutyrate (PdBU), interleukin 1 or interleukin 2 . The precise lineage and in vivo function of decidual granulated lymphocytes remains to be established.

Cancer Lett, 1989 Aug, 46(3), 213 - 9
Prognostic significance of DNA content in large bowel carcinoma: a retrospective flow cytometric study; Scivetti P et al.; Flow cytometric (FCM) determination of DNA content was performed on surgical specimens from 44 patients with previously untreated colonic carcinoma . For each tumor, cell suspensions were prepared from 2-4 40-microns thick sections obtained from formalin-fixed and paraffin embedded tissue samples . Aneuploidy was found in 47.2% of all the tumors and the aneuploid clone had a median DNA index of 1.49 (range: 1.24-1.93) . Aneuploidy was found in 26.7% of highly differentiated tumors (WHO histologic classification), in 53.8% of moderately differentiated tumors and in 100% of poorly differentiated tumors (P = 0.04) . The 33.3% of stages 1 + 2 (TNM) and the 70.6% of stages 3 + 4 tumors were aneuploid (P = 0.002) . Median survival from surgery was 46.4 months for all patients . It was 18.8 months for patients with aneuploid tumors and 85.7 months for those with diploid tumors (P = 0.0002) . FCM determination of DNA in colon carcinomas can easily be performed on archival histological material and provides prognostic information.

J Pharmacol Exp Ther, 1989 Aug, 250(2), 632 - 6
Contribution of the intestine to the first-pass metabolism of felodipine in the rat; Wang SX et al.; The systemic availability of intraduodenally (i.d.) administered felodipine in the rat is about 10% . The purpose of this study was to determine to what extent intestinal metabolism contributes to the first-pass elimination of felodipine in the rat . Four different types of experiments were performed . 1) {3H}Felodipine was given i.v . and i.p.; 2) the uptake of i.p . administered {3H}felodipine by the lymph was studied for 3 hr after dosing; 3) portal blood was collected quantitatively for 40 min after i.d . administration of {3H}felodipine; and 4) the in vitro metabolism of felodipine was studied in intestinal cell suspensions . The mean bioavailability of the i.p . dose was approximately 48% . The uptake via the lymph was negligible as an insignificant amount of the radioactive i.p . dose was recovered in lymph from a main lymph vessel in the peritoneal cavity . An average of 21 +/- 12% of given radioactive dose was recovered in portal blood during the first 40 min after i.d . dosing . The recovered radioactivity was to 40 to 70% due to felodipine and 9 to 16% was due to dehydro-felodipine . These results indicate that substantial first-pass elimination occurs in the intestine of the rat . Further support for gastrointestinal metabolism of felodipine in the rat was obtained from incubations with intestinal cells.

J Neurochem, 1989 Aug, 53(2), 482 - 8
Restoration of high affinity choline uptake in the hippocampal formation following septal cell suspension transplants in rats with fimbria-fornix lesions; Kaseda Y et al.; High affinity choline uptake (HACU) was investigated in the hippocampal formation following fetal septal cell suspension transplants into rats with fimbria-fornix lesions . Nine-14 weeks after transplantation, HACU was markedly decreased in hippocampi from animals with fimbria-fornix lesions; this decrease was ameliorated by fetal septal cells transplanted into the host hippocampus . HACU related to septal transplantation was activated in vitro by K+, and in vivo by the administration of scopolamine and picrotoxin . These findings suggest that fetal septal cell transplantation can restore HACU in the host hippocampus following fimbria-fornix lesions, and that HACU related to the graft has pharmacological properties similar to those of the normal adult HACU system . The activation of HACU by picrotoxin, a gamma-aminobutyric acid (GABA) antagonist, suggests that transplanted cholinergic neurons receive either direct or indirect functional input from GABAergic afferents from the transplant and/or host hippocampus . Lesions of the fimbria-fornix also resulted in an increased binding to muscarinic receptors in the dorsal hippocampus . This increase in binding was not significantly ameliorated by intrahippocampal grafts of cholinergic neurons.

Can J Physiol Pharmacol, 1989 Aug, 67(8), 820 - 8
Nuclear magnetic resonance monitoring of sodium in biological tissues; Boulanger Y et al.; In recent year, the 23Na nuclear magnetic resonance (NMR) technique has been applied to the study of biological tissues . The advantages of this method are noninvasiveness and good sensitivity . The resonances of the intra- and extra-cellular sodium can be separated by the addition of shift reagents to the extracellular compartment . The method has been mostly applied to cell suspensions, kidney tubules, glands, and small organs . Owing to line-broadening effects, the NMR visibility of the intracellular sodium is reduced to 40% in most cases but can be lower or higher . Time-dependent measurements are possible with adequate life-supporting equipment, allowing the determination of transport parameters . 23Na relaxation times are short in tissues (below 50 ms) and highly dependent on the medium composition . The application of the 23Na NMR technique to intact organs can be hampered by the difficulty of getting a good distribution of the shift reagent in the extracellular milieu . A summary of the studies performed is presented with specific examples to illustrate typical applications.

Zh Mikrobiol Epidemiol Immunobiol, 1989 Aug, (8), 14 - 7
{Observation of the processes of low-temperature crystallization in an aqueous cell suspension by electron microscopy}; Sventitskii EN et al.; The possibility of using electron microscopy for the study of the processes of the low-temperature crystallization of water in cell suspension is discussed with E . coli M-17 culture used as an example . The replacement of water in the frozen state permits the observation of processes occurring in the sample at low temperature . The advantages of the method are demonstrated in the study of objects previously frozen at different rates.

Am J Surg Pathol, 1989 Aug, 13(8), 691 - 9
Multiple lymphomatous polyposis of the gastrointestinal tract . A clinicopathologically distinctive form of non-Hodgkin's lymphoma of B-cell centrocytic type; O'Briain DS et al.; Multiple lymphomatous polyposis of the gastrointestinal tract was initially described as mucosal lymphomatous involvement by any of a variety of Hodgkin's or non-Hodgkin's lymphomas that produced a polypoid appearance over long segments of the gastrointestinal tract . We studied four patients in whom histology revealed diffuse small cleaved cell lymphoma (one case), or intermediate lymphocytic lymphoma of diffuse type (one case), or mantle zone pattern (two cases) . All four cases are classifiable as centrocytic lymphoma . Cell suspension and immunocytochemical studies demonstrated B-cells of IgMD or M type with light chain restriction (two kappa, two lambda) showing a B1+ HLA Dr+ LN2+ CD5+ CD10+ . Although all four patients had a partial response to combination chemotherapy, three of them died within 3 years . Analysis of 24 cases reported since 1971 (including the present cases) suggests that MLP is a distinct clinicopathological entity that results from gastrointestinal involvement by a B-cell centrocytic lymphoma . It is distinct from the recently described clinicopathological forms of centrocytic lymphoma and intermediate lymphocytic lymphoma, which both show extensive peripheral lymphadenopathy and splenomegaly, but it is probably closely related to them . The differences are probably attributable to distinct cell tropism or homing properties rather than to cellular histogenesis or degree of maturation.

Differentiation, 1989 Aug, 41(2), 127 - 38
Isolation of subpopulations of murine epidermal cells using monoclonal antibodies against differentiation-related cell surface molecules; Mackenzie IC et al.; Monoclonal antibodies (mAbs) against epithelial cells were prepared by immunization of rats with lyophilized murine epithelia . Screening against tissue sections and epithelial cell suspensions permitted identification of mAbs against surface molecules that are expressed early in cell differentiation . Staining with these mAbs followed by fluorescence-activated cell sorting enabled isolation of subpopulations of basal epithelial cells . Staining these subpopulations with antibodies against known differentiation markers (cytokeratins and bullous pemphigoid antigen) and measurements of cell size indicated that they represented fractions of the basal cell population in sequential stages of early differentiation . Labeling mice with bromodeoxyuridine at various times prior to cell isolation showed that the least-differentiated basal cells cycle more slowly than those at later stages, data which support the concept of a differentiation-related, hierarchical pattern of organization of the proliferative compartment.

J Dermatol, 1989 Aug, 16(4), 303 - 7
Flow cytometric analysis of murine splenic cells after DNFB painting on Langerhans cell deficient skin; Morita H et al.; One week after a single painting of 50 microliters of 0.5% DNFB in acetone-olive oil on the tail skin of C3H mice, the spleens were removed from the animals . A single cell suspension was prepared from the spleens, and flow cytometric analysis was performed for L3T4 and Lyt2 positive cells, as well as for the IJk expression of the Lyt2+ cells . Results showed that the percentages and absolute numbers of Lyt2+ L3T4- cells, Lyt2- L3T4+ cells, Lyt2+ IJk+ cells, and Lyt2+ IJk- cells in the spleens of the DNFB-treated mice were not significantly different from those in the non-treated mice . However, in the treated mice, the IJk expression of Lyt2+ cells intensified . These results indicate that, following a single painting of DNFB onto Langerhans cell-deficient skin, the numbers of Lyt2+ cells do not change significantly, but do change functionally.

Eur J Immunol, 1989 Aug, 19(8), 1513 - 6
Murine enterocytes can present soluble antigen to specific class II-restricted CD4+ T cells; Kaiserlian D et al.; Highly purified mature epithelial cells (EC) from murine small intestinal villi (excluding Peyer's patch epithelium) were examined for their capacity to present foreign antigen to T cells . Cell suspensions composed of 99% Ia+T200- EC were able to present keyhole limpet hemocyanin to an antigen-specific class II-restricted L3T4+ T cell hybridoma, and stimulate interleukin 2 production . Antigen presentation by EC was inhibited by major histocompatibility complex class II-specific monoclonal antibodies . It is concluded that EC express functional class II molecules and may activate adjacent CD4+ T cells to induce lymphokine synthesis.

Am J Clin Pathol, 1989 Aug, 92(2), 206 - 9
Simultaneous determination of absolute total lymphocyte and CD4+ lymphocyte levels in peripheral blood by flow cytometry; Prince HE et al.; A distinguishing feature of the Spectrum III flow cytometer is its capacity to analyze a constant volume of cell suspension . The authors have capitalized on this feature to directly and simultaneously measure the absolute numbers of all lymphocytes and CD4+ lymphocytes per microliter of peripheral blood . The study group consisted of 42 hospital patients, 12 former blood donors seropositive for human immunodeficiency virus (HIV), and 12 HIV-seronegative donors . Regression analysis revealed a highly significant correlation (r = 0.97) between Spectrum lymphocyte counts and lymphocyte counts determined by a Coulter Counter S-Plus IV . Similarly, Spectrum CD4 cell counts were significantly correlated (r = 0.98) with CD4 cell counts calculated from the Coulter lymphocyte counts and % CD4+ lymphocytes determined by flow cytometry . These findings indicate that the absolute numbers of lymphocytes and subsets of lymphocytes in peripheral blood can be rapidly and simultaneously measured by flow cytometry . Such an assay should prove useful in studies of HIV infection, where total lymphocyte and CD4 cell levels are important parameters for clinical staging and assessing responses to treatment.

J Gen Physiol, 1989 Aug, 94(2), 385 - 400
Glutaraldehyde fixation of the cAMP-dependent Na+/H+ exchanger in trout red cells; Motais R et al.; It has been shown that the addition of a beta-adrenergic catecholamine to a trout red blood cell suspension induces a 60-100-fold increase of sodium permeability resulting from the activation of a cAMP-dependent Na+/H+ antiport . Subsequent addition of propranolol almost instantaneously reduces the intracellular cAMP concentration, and thus the Na permeability, to their basal values (Mahe et al., 1985) . If glutaraldehyde (0.06-0.1%) is added when the Na+/H+ exchanger is activated after hormonal stimulation, addition of propranolol no longer inhibits Na permeability: once activated and fixed by glutaraldehyde, the cAMP dependence disappears . Glutaraldehyde alone causes a rapid decrease in the cellular cAMP concentration . In its fixed state the antiporter is fully amiloride sensitive . The switching on of the Na+/H+ exchange by cAMP is rapidly (2 min) followed by acute but progressive desensitization of the exchanger (Garcia-Romeu et al., 1988) . The desensitization depends on the concentration of external sodium, being maximal at a normal Na concentration (145 mM) and nonexistent at a low Na concentration (20 mM) . If glutaraldehyde is added after activation in nondesensitizing conditions (20 mM Na), transfer to a Na-rich medium induces only a very slight desensitization: thus the fixative can "freeze" the exchanger in the nondesensitizing conformation . NO3- inhibits the activity of the cAMP-dependent Na+/H+ antiporter of the trout red blood cell (Borgese et al., 1986) . If glutaraldehyde is added when the cells are activated by cAMP in a chloride-containing medium, the activity of the exchanger is no longer inhibited when Cl- is replaced by NO3- . Conversely, after fixation in NO3- medium replacement of NO3- by Cl- has very little stimulatory effect . This indicates that the anion dependence is not a specific requirement for the exchange process but that the anion environment is critical for the switching on of the Na+/H+ exchanger and for the maintenance of its activated configuration.

Am J Physiol, 1989 Aug, 257(2 Pt 1), C323 - 32
Continuous membrane voltage recordings in A10 vascular smooth muscle cells: effect of AVP; Korbmacher C et al.; Continuous membrane voltage (V) recordings were obtained in A10 vascular smooth muscle cells (rat aorta) using glass microelectrodes . Resting membrane voltage in 262 impalements averaged 54.0 +/- 0.4 (SE) mV . Relative K+ conductance was characterized, and the contribution of electrogenic Na+-K+-ATPase to membrane voltage was investigated . Action potentials could be induced by application of 1 mM barium or 10(-4) M acetylcholine . In a few recordings, spontaneous spike activity occurred, and this could be abolished by 5 mM MgCl2 or by removal of extracellular Ca2+ . Barium-induced action potentials were not dependent on the presence of extracellular Na+ and not inhibitable by 10(-6) M tetrodotoxin . Application of 10(-6) M {Arg8} vasopressin (AVP) for 30 s caused a typical biphasic membrane voltage response with an initial transient hyperpolarization of -9.5 +/- 1.1 mV and a more sustained subsequent depolarizing response averaging 28.2 +/- 1.3 mV (mean +/- SE, n = 58) . The effect of AVP on membrane voltage was blocked by the V1-antagonist {beta-mercapto-beta,beta-cyclopentamethylenepropionyl1,O-Me- Tyr2,Arg8}vasopressin . The initial hyperpolarizing component of the membrane voltage response to AVP became more prominent when V was predepolarized, for example, by a preceding AVP application . However, when AVP was applied during high K+ depolarization or in the presence of quinidine (1 mM), the initial hyperpolarizing response was practically abolished . The time course of the initial hyperpolarization was shown to be similar to the calcium transient observed in fura-2-loaded A10 cell suspensions after the application of AVP . We conclude that the initial AVP-induced hyperpolarization in A10 cells corresponds to an activation of Ca2+-activated K+ channels.

J Clin Endocrinol Metab, 1989 Aug, 69(2), 280 - 6
Angiotensin II stimulates both inositol phosphate production and human placental lactogen release from human trophoblastic cells; Petit A et al.; We studied the functional significance of the binding of angiotensin-II (AII) to human placentas . Human trophoblastic cell suspensions were prepared by trypsin digestion of minced tissue . Cell incubations with increasing doses of {125I}(SAR1)AII, ranging from 0.01-2.5 nmol/L, were carried out for 20 min at 37 C . The results indicated the presence of specific low capacity {4300 +/- 1300 (+/- SE) sites/cell}, high affinity (Kd = 0.38 +/- 0.06 nmol/L) binding sites for {125I}(Sar1)AII . This binding was specific for AII analogs . When placental cells were preloaded with 40 microCi/mL {3H}myoinositol for 2 h at 37 C, AII stimulation resulted in a dose-dependent increase in inositol phosphate (InsP) production {EC50 = 1.4 +/- 0.4 (+/- SE) nmol/L}, as measured by ion exchange chromatography . (Sar1)AII also stimulated InsP production, with an EC50 of 0.3 +/- 0.2 nmol/L . AII-stimulated production of InsP was completely blocked by the antagonist (Sar1,Ala8)AII . AII also stimulated human placental lactogen release from trophoblastic cells in a dose-dependent fashion . The EC50 was 18 +/- 9 pmol/L, and the stimulation was blocked by (Sar1,Ala8)AII, as found for AII-stimulated InsP production . These results suggest that stimulation of human placental lactogen release by AII may be mediated by activation of phospholipase-C, which, in turn, produces phosphoinositide breakdown . The results, therefore, provide evidence of a physiological role for the renin-angiotensin system within the human placenta.

Photochem Photobiol, 1989 Aug, 50(2), 175 - 83
Singlet oxygen generation of porphyrins, chlorins, and phthalocyanines; Kimel S et al.; The production of singlet oxygen was measured indirectly for three classes of photosensitizers: porphyrins (Photofrin II, TPPS4), chlorins (MACE, DACE), and a phthalocyanine (CASPc) . Buffered solutions of sensitizers and singlet oxygen acceptors were irradiated with a CW dye laser and the oxygen depletion was monitored electrochemically with a Clark-type microelectrode . A comparison of oxygen-depletion rate constants and quantum efficiencies yields the order of efficiency of the sensitizers: TPPS4 greater than MACE greater than PII greater than DACE greater than CASPc . For singlet oxygen acceptors the order was: furfuryl alcohol greater than imidazole greater than tryptophan . CHO cell suspensions were also used as acceptors . Here the order of efficiency (per absorbed photon) was PII greater than MACE approximately CASPc . Expressed in terms of oxygen depletion per cell the order was CASPc approximately PII greater than MACE . When performing cell clonogenicity studies the order of efficiencies, expressed as percentage cell kill per unit weight of sensitizer, was CASPc greater than PII greater than MACE approximately DACE . The discrepancy between the efficiencies of sensitizers to generate singlet oxygen and their cytotoxicity was explained in terms of photodegradation (for the chlorins), intracellular localization (for PII), and contributions from a Type I mechanism (for CASPc).

J Interferon Res, 1989 Aug, 9(4), 475 - 89
Effect of human interferon-alpha and interferon-gamma on growth, histology, and DNA content of human osteosarcomas in nude mice; Brosjo O et al.; The antitumor effect of human natural and recombinant interferon-gamma (IFN-gamma) was evaluated in human osteosarcomas grown as xenografts in nude mice . IFN-gamma was given as daily subcutaneous injections, alone or in combination with IFN-alpha . The growth of two out of three tested osteosarcomas was inhibited by 2 x 10(5) IU of natural IFN-gamma . A five times higher dose of recombinant IFN-gamma, as compared with natural (n) IFN-gamma, was needed to obtain growth inhibition of one osteosarcoma . This difference in dose-response could be explained by differences in pharmacokinetics . Hence, subcutaneously administered natural IFN-gamma gave 10 times higher serum levels than obtained with the recombinant type . Combination treatment with IFN-alpha and IFN-gamma induced a potentiation of the antitumor effect in one osteosarcoma . In another osteosarcoma, 2-4 x 10(5) IU of nIFN-gamma did not effect tumor growth and could not potentiate the antitumor effect of 2-4 x 10(5) IU of nIFN-alpha . By using DNA analysis in cell suspension and tissue section, the proportion of aneuploid tumor cells within the xenograft could be estimated . This analysis showed that the antitumor effects of IFN were more pronounced than mere measurement of tumor volume suggested . IFN-inhibited tumors were partly replaced by fibroblasts or bone tissue . In conclusion, at the doses given nIFN-gamma appeared to have similar antitumor effects as IFN-alpha in two osteosarcomas, whereas one was sensitive to only IFN-alpha . Combination IFN treatment induced a potentiation of the antitumor effect in one osteosarcoma but not in another . The differences between the osteosarcomas in obtained antitumor effect of IFN treatment probably reflects individual IFN sensitivity and demonstrates the importance of assessing several tumors of the same neoplastic entity.

Brain Res Dev Brain Res, 1989 Aug 1, 48(2), 215 - 28
Vascularization of fetal cell suspension grafts in the excitotoxically lesioned adult rat thalamus; Dusart I et al.; Several studies have considered the establishment of vascularization in intracerebral solid transplants of neural tissue . The widely supported interpretation of the results is that the vascular network of the solid grafts is already present before implantation into the host brain . The situation is different when dissociated fetal tissue is transplanted as a cell suspension because in these conditions the fetal vascular network is disrupted . The present study has, therefore, been undertaken to follow the angiogenesis in a transplant of dissociated fetal cells implanted into the excitotoxically neuron-depleted thalamus . The vascular network is compared to that observed in the intact and in the lesioned thalamus both in terms of morphology of the capillaries and of the function of the blood-brain barrier (BBB) . In the transplant, capillaries, stained by Indian ink, are very few in number and have very fine calibers during the first 20 days after grafting . Some structures can be identified as immature blood vessels at the electron microscopic level . The blood vessels are progressively more numerous in the graft and they demonstrate mature ultrastructural features 2 months after grafting . Last, there is no leakage of the BBB for peroxidase . The vascularization seems to follow a pattern of maturation comparable to that described during development in the literature . In contrast, in the lesioned area, there is a reactive angiogenesis: 10 days after the excitotoxic injection (shortest time studied), there are many wide caliber vessels with expanded perivascular spaces engorged with mesodermal cells . A microvascularization also develops transiently during the first two months . Capillaries are abnormal from the functional point of view, since there is a leakage of the BBB to macromolecules . The use of an experimental model in which transplant had to grow in a lesioned area permits to determine two types of vascularization: an apparently normal developmental timetable, normal morphological and functional characteristics, in the transplant; a reactive angiogenesis, in the lesioned area.

Am J Physiol, 1989 Aug, 257(2 Pt 1), C197 - 206
Ca2+ induces charybdotoxin-sensitive membrane potential changes in rat lymphocytes; Grinstein S et al.; There is disagreement regarding the existence of Ca2+-activated K+ channels in lymphocytes . Depolarization, hyperpolarization, or little change in membrane potential (Em) has been reported following elevation of free cytosolic Ca2+ concentration ({Ca2+}i) . Patch-clamping studies have demonstrated inhibition of voltage-gated K+ channels, but Ca2+-activated K+ channels have not been detected . We used charybdotoxin (CTX), a potent inhibitor of Ca2+-activated K+ channels, to assess their presence in rat thymic lymphocytes . Fluorescent probes were used to measure Em and {Ca2+}i in cell suspensions treated with ionomycin . At basal {Ca2+}i, CTX had no effect on Em, suggesting that Ca2+-activated K+ channels do not contribute importantly to the resting potential . Elevation of {Ca2+}i in the submicromolar range induced a hyperpolarization that was dependent on the outward K+ gradient . The shape and duration of the Em change closely followed the elevation of {Ca2+}i . This hyperpolarization was inhibited by nanomolar concentrations of CTX . When {Ca2+}i approached or exceeded 1 microM, a biphasic Em change was recorded . A transient, CTX-sensitive hyperpolarization was followed by a sustained depolarization . The latter was greatly reduced when external Na+ was omitted . The data suggest that thymic lymphocytes possess Ca2+-sensitive K+ channels, which are activated by moderate increases in {Ca2+}i, resulting in hyperpolarization . At higher {Ca2+}i, the effect of K+ channels on Em is superseded by opening of nonselective cation channels, producing depolarization . Variations in the level of {Ca2+}i attained in earlier studies can explain existing discrepancies.

Biochim Biophys Acta, 1989 Jul 24, 983(1), 42 - 50
Enhanced hybridoma production by electrofusion in strongly hypo-osmolar solutions; Schmitt JJ et al.; Electrofusion of mammalian cells in strongly hypo-osmolar media containing sorbitol, small amounts of divalent cations and albumin resulted in high yields of hybrids . The number of viable hybrids was higher than any value for chemically- or electrically-mediated fusion reported in the literature . Optimum clone numbers were obtained for fusion of osmotically-stable subclones of murine myeloma cells with DNP-Hy-stimulated lymphocytes provided that the osmolarity of the fusion medium was as low as 75 mosmol/l . Similar results were obtained for fusion of osmotically stable subclones of myeloma cells with the murine hybridoma cell line G8 . Due to the dramatic increase in volume the field strength of the breakdown pulse (leading to fusion of the dielectrophoretically aligned cells) has to be reduced, as predicted by theory . The efficacy of hypo-osmolar electrofusion allowed the use of very few cells (about 10(5) lymphocytes or G8 cells per fusion chamber) . This figure is considerably smaller than that reported in the literature for iso-osmolar electrofusion . It is significant that, in contrast to iso-osmolar conditions, the fusion yield in hypo-osmolar electrofusion was reproducible over long periods of time and less dependent of variations between cultures . At suspension densities of about 10(6) cells per fusion chamber (normally used in iso-osmolar electrofusion) hypo-osmolar electrofusion of homogeneous cell suspensions resulted in the formation of many giant cells when the appropriate field conditions were applied . Similar high or, at some field strengths, even higher numbers of clones at low cell suspension density were obtained when G8 and myeloma cells were first exposed during the washing procedure to strongly hypo-osmolar media, but then transferred to iso-osmolar solutions for electrofusion . Similar experiments with lymphocytes and myeloma cells failed because of destruction of many lymphocytes by the two osmotic shock steps in rapid succession . Volume distribution measurements of G8 and myeloma cells showed that after re-incubation of the osmotically pre-stressed cells the original volume distribution is largely, but not completely re-established . This and other results indicate that osmotic pressure gradients and associated tensions in the membrane do not play a primary role in the initiation of the electrofusion process . The experiments suggest that due to the osmotic (pre-) stress the membrane permeability is slightly and uniformly increased presumably due to the dissolution of membrane- and cell-skeleton proteins . Obviously, this facilitates electrofusion in hypo-osmolar or subsequently in iso-osmolar solutions.

Toxicology, 1989 Jul 17, 57(2), 217 - 23
Formation of nuclear anomalies in rat intestine by benzidine and its biliary metabolites; Percy AJ et al.; Administration of benzidine (BZ) by intraperitoneal injection (i.p.) to rats (0-100 mg/kg) produced, after 24 h, a dose-dependent formation of nuclear anomalies (micronuclei, pyknotic and karyorrhectic nuclei) in intestinal epithelial cells analysed both in isolated cell suspensions and in the intestinal crypts in tissue sections . When bile collected (0-4 h) from rats treated with BZ (150 mg/kg, i.p.) was infused into the duodenum of recipient rats, nuclear anomalies were observed in mucosal epithelial cells, after 24 h, with a similar distribution to that in rats given BZ by i.p . injection . The formation of nuclear anomalies in the intestine is in accord with the intestinal carcinogenic effect of BZ and is, at least partially, dependent on exposure of epithelial cells to biliary metabolites of BZ.

Biochem Biophys Res Commun, 1989 Jul 14, 162(1), 9 - 14
Intracellular protein phosphorylation in oat (Avena sativa L.) protoplasts by phytochrome action . 1 . Measurement of action spectra for the protein phosphorylation; Park MH et al.; The effects of red light and wavelength dependency of the protein phosphorylation in oat protoplasts were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography . Red light (660 nm) irradiation of the protoplasts increased the phosphorylation of 15 different proteins, and the phosphorylation of 2 proteins (27 KDa, 32 KDa) out of 15 were observed to be dependent on the wavelength of the irradiating light . The phosphorylation densities of these two proteins increased up to two or three hundred percent during a three-minute period of irradiation . The phosphorylation of these two proteins revealed a red/far-red photoreversibility of phytochrome . When a calcium ion chelator (2 mM EGTA) was added into the cell suspension, the phosphorylations of all the proteins were reduced about 200% . These findings suggest that phytochrome action and Ca2+ influx are certainly involved in the in vivo phosphorylation of proteins in oat protoplasts.

Am J Physiol, 1989 Jul, 257(1 Pt 1), C65 - 76
Cytoplasmic pH in pulmonary macrophages: recovery from acid load is Na+ independent and NEM sensitive; Bidani A et al.; The pulmonary macrophage plays a primary role in the immunological defense of the lung . Although many studies have been devoted to elucidation of its phagocytic and secretory functions, little is known of its membrane transport properties or of how it regulates intracellular pH (pHi) . The purpose of this study, therefore, was to determine base-line pHi and the mechanism(s) by which the cell recovers pHi when challenged with an intracellular acid load . Through the use of the pH-sensitive fluorescent dye, 2,7-biscarboxyethyl-5(6)-carboxy-fluorescein (BCECF), base-line pHi was estimated to be 7.24 +/- 0.03 . Cells were acidified by two methods, nigericin and weak acids, while recovery (dpHi/dt) was monitored . The rate of recovery was found to be independent of external Na+ and K+ and was insensitive to amiloride . Pretreatment with 4,4'-diiso-thiocyanatostilbene-2,2'-disulfonic acid, an inhibitor of Cl- -HCO3- exchange, was also without effect on recovery from an intracellular acid load in these cells, under nominally HCO3- -free conditions . In contrast, N-ethylmaleimide (NEM) and N,N'-dicyclohexylcarbodiimide, nonspecific inhibitors of proton adenosinetriphosphatases (ATPases), virtually abolished pHi recovery . Efflux of H+ equivalents by pulmonary macrophages was measured by techniques involving both pH stat titration and the effect on fluorescence of extracellular BCECF . Basal H+ extrusion was approximately 2.75 +/- 0.64 nmol H+.min-1.10(6) cells-1 and was enhanced to approximately 26.0 +/- 6.95 nmol H+.min-1.10(6) cells-1 in acid-loaded cell suspensions . The basal rate of H+ extrusion was reduced to approximately 0.84 +/- 0.31 nmol H+.min-1.10(6) cells-1 in the presence of 1 mM NEM . These results suggest that recovery of cytoplasmic pH from an intracellular acid load, as well as regulation of pHi, under the conditions examined, is not mediated by a Na+-H+ exchanger in these cells . Rather, the data are consistent with the presence of an H+-ATPase in the plasma membrane of pulmonary macrophages.

Brain Res, 1989 Jul 10, 491(2), 205 - 11
Rapid migration of grafted cortical astrocytes from suspension grafts placed in host thoracic spinal cord; Bernstein JJ et al.; The cerebral cortices from 14-day gestation rat embryos were prelabeled with Phaseolus vulgaris leucoagglutin (PHAL) and homografted as a cell suspension into host thoracic spinal cord . Animals were sacrificed at 7, 14, 21 and 30 days postimplantation (DPI) . Paraffin sections of cervical, thoracic and lumbar spinal cord were double-labeled for the presence of glial fibrillary acidic protein (GFAP) a specific marker for astrocytes, and PHAL, utilized as a marker for graft-derived cells . PHAL-GFAP positive cells were found throughout the thoracic spinal cord at all time periods, indicating that grafted astrocytes migrated along all 3 axes of the host spinal cord (rostral-caudal, dorsal-ventral and left-right) . At 30 DPI, graft-derived astrocytes were found in host cervical and lumbar spinal cord . There appeared to be a minimal delay in the onset of migration.

Anticancer Res, 1989 Jul-Aug, 9(4), 941 - 6
Treatment of mice bearing a Krebs ascitic tumor by means of a protocol based on radioactive copper (64Cu) . I . The "cancer animal": a new concept leading to an effective antitumoral treatment; Apelgot S et al.; Previous experiments have shown that 64Cu transmutation was inefficient to cure mice which had been injected 6 days earlier with 5 x 10(5) Krebs ascitic cells . The experiments were repeated with 64CuCl2 administered only 1 day after the injection of 5 x 10(5) Krebs ascitic cells when no developing tumor existed . The results reported showed that, even in this case, only a delay in death could be observed . These negative results revealed that malignant cells injected 24 hours previously in a mouse (in vivo conditions) differ from a malignant cell suspension in a tube (in vitro conditions) where the lethal effect of 64Cu transmutation was clearly evidenced . We concluded that some kind of collaboration was established between the few accepted malignant cells and the host . Based on this collaboration we introduced a new concept, "the cancer mouse", in which in addition to the malignant cells some cells in the host, even though non - malignant, nonetheless feature a slightly modified functioning: "the cancer functioning" . In this view, an efficient antitumor treatment must act at the same time and in a coordinated manner on the malignant cells and on the host cells which now feature "the cancer functioning" . In the efficient treatment described (Anticancer Res 9: 947-954, 1989) some compounds (64Cu and thioproline) act against the malignant genomes, and other components (metal ions, amino acids, vitamin D2, thyroxine and chelating substances) act against the functioning of the host cells by taking into account certain characteristics of living systems . This treatment was efficient in curing mice injected 1 or 6 days previously with 5 x 10(5) Krebs ascitic cells.

Respir Physiol, 1989 Jul, 77(1), 31 - 9
Gas transfer in isolated lungs perfused with red cell suspension or hemoglobin solution; Geiser J et al.; Rapid mixing experiments have shown that the reaction between oxygen and hemoglobin is faster in hemoglobin solutions than in red cell suspensions . In this study we tested whether this observation can also be made in the lung . Excised rabbit lungs were perfused either with washed human red cell suspensions or with hemoglobin solutions, each with 50 g hemoglobin/L, and steady-state diffusing capacities (DLO2) for oxygen elimination measured . Mean settings were a temperature of 29.5 degrees C of the inflowing and outflowing perfusate of the lung, a total ventilation of 1.7 L.min-1, and a perfusion rate of 116 ml.min-1 . Under those conditions resulted a DLO2 with hemoglobin solution of 0.68 +/- 0.18 ml.min-1.mm Hg-1, and a significantly lower value of 0.50 +/- 0.06 ml.min-1.mm Hg-1 with red cell suspension (P less than 0.01) . An extraerythrocytic diffusing resistance, formed by a plasma layer and/or arising from a dynamic diffusion boundary layer, which is also known as unstirred layer, could explain the lower value with red cell suspensions.

Cell Immunol, 1989 Jul, 121(2), 225 - 36
Inhibition of murine lymphokine-activated killer (LAK) cell activity by adherent cells; Longley RE et al.; The effects of adherent cell depletion, indomethacin, and prostaglandin E2 (PGE2) on murine LAK cell activity were investigated . Removal of plastic adherent cells from splenocyte suspensions either prior to 5-day culture with 1000 U/ml of recombinant human IL-2 (rIL-2) or prior to assay resulted in an enhanced LAK cell cytotoxicity compared to that of whole spleen cell suspensions . Indomethacin enhanced LAK cell cytotoxicity of whole splenocyte suspensions if present during the culture period, but had no effect on whole splenocyte or adherent cell-depleted cell suspensions if added just prior to assay . PGE2 suppressed LAK cell activity of nonadherent splenocyte but not whole splenocyte suspensions when present during the culture period . In vivo treatment of mice with indomethacin enhanced cytotoxicity directed toward both LAK sensitive, natural killer (NK) resistant (P-815) and LAK, NK sensitive (YAC-1) tumor cell targets . Splenocytes from indomethacin-treated mice cultured with additional indomethacin and rIL-2 exhibited highest LAK cell activity . The results from this study indicate that LAK cells are regulated by adherent cells which suppress LAK cell activity . This suppression can be reversed both in vitro and in vivo by indomethacin . This study has important implications for the possible clinical use of indomethacin in the potentiation of in vivo and in vitro LAK cell activity for immunotherapeutic protocols.

Neuropathol Appl Neurobiol, 1989 Jul-Aug, 15(4), 331 - 8
Prognosis in malignant glioma: a retrospective study of biopsy specimens by flow cytometry; Jimenez O et al.; The DNA ploidy of a series of 78 gliomas has been estimated by flow cytometry using cell suspensions prepared from paraffin embedded material . Apart from two oligodendrogliomas the tumours were all astrocytomas . Forty-nine (63%) tumours were found to have a diploid DNA distribution and 29 (37%) an aneuploid DNA distribution . The two oligodendrogliomas were both DNA aneuploid . No correlation was found between DNA ploidy and histological grade or DNA ploidy and survival . Both ploidy groups appear to have responded equally to therapy . In this study the factors associated with a short survival were advancing age and the presence of vascular endothelial proliferation, while the DNA ploidy and the cytological features do not appear as useful in predicting survival when examining biopsy material.

Int J Cell Cloning, 1989 Jul, 7(4), 232 - 41
Comparison between clonogenic and cytotoxic assays for measuring LAK cell activity; Kluin-Nelemans HC et al.; The antiproliferative effect of lymphokine-activated killer (LAK) cells was studied using a clonogenic assay in an attempt to find a model for predicting this effect in vivo or ex vivo (in the case of purging) in cancer treatment . The results were compared with the standard 51Cr-release cytotoxic assay . Cells from clonogenic neoplastic cell lines (K562 and HL-60) were plated in methylcellulose with LAK cells obtained from ten different donors in various effector-to-target (E:T) ratios . At E:T ratios of 16:1, elimination of greater than 90% of the clonogenic cells was seen in 20 of 21 experiments, whereas such lysis was incidentally found in the 51Cr-release assay . In almost all paired combinations, clonogenic cells tested in a colony assay were more sensitive to kill by LAK cells than the whole tumor cell suspensions measured in the 51Cr-release assay.

Cytometry, 1989 Jul, 10(4), 442 - 7
Accuracy of routine flow-cytometric bitmap selection for three leukocyte populations; Sucic M et al.; A double-blind study was performed with peripheral blood of 41 human subjects to check the accuracy of determination of lymphocyte, monocyte, and granulocyte windows with which every flow cytometric analysis of leukocyte markers starts . White blood cell suspensions were prepared according to the whole blood method and analyzed on an EPICS-C flow cytometer using the two-parameter 90 degrees light scatter vs . forward angle light scatter (granularity vs . cell size) data distribution . Windows (bitmaps) for lymphocytes, monocytes, and granulocytes were drawn and numbers of cells determined in each . The proportions of lymphocytes, monocytes, and granulocytes were calculated in relation to total cell number, counted and in relation to the sum of cells in three bitmaps, and then compared with proportions determined by microscopic whole blood cell (WBC) differential and a WBC differential determined in an automated hematology analyzer . Average proportions of lymphocytes obtained by the flow cytometer were significantly lower than those obtained by either microscopic or automated differential, suggesting that some of the relevant cells were not included in the bitmaps . Granulocyte proportion related to total cell number was lower and that related to bitmap cell number higher than that obtained by microscopic and automatic differentials, suggesting that nongranulocytic cells were included in the granulocyte bitmaps . Proportions of lymphocytes and granulocytes obtained by the flow cytometer correlated well with those obtained by both microscopic and automatic differential . In contrast, the proportions of monocytes showed a poor correlation, which is probably due to their low number and delicate position in the distribution, and which makes them difficult to delineate.

Acta Otolaryngol, 1989 Jul-Aug, 108(1-2), 152 - 60
The rabbit VX2 tumour as a model for carcinomas of the tongue and larynx; Jefferis AF et al.; Animal models of carcinomas can provide useful information for the evaluation of new treatments . In the rabbit, the VX2 tumour is a fast growing, transplantable squamous cell carcinoma . By implanting a cell suspension of the tumour into the tongue and/or the larynx, a useful new model for studying or treating tumours in this region has been developed . The tumours were evaluated histologically and showed features consistent with human carcinomas, including surface ulceration, lymphatic spread and encroachment of the airway . The VX2 tumours in these sites have features which offer advantages over existing models . These include the ability to study several sites of tumour growth in the same animal, to follow rapidly growing tumours, storage of the tumour in liquid nitrogen allowing experiments to be started when convenient, and the large size of the rabbit larynx which permits considerable tumour growth before airway embarrassment.

J Reprod Fertil, 1989 Jul, 86(2), 759 - 66
Isolation of rat gonocytes by velocity sedimentation at unit gravity; van Dissel-Emiliani FM et al.; A method for obtaining enriched populations of gonocytes from rat embryos at 18 days p.c . has been developed . Single cell suspensions with high cell yield and good viability of the cells were obtained by a collagenase/trypsin digestion of the testes . Cells were separated on the basis of size by the Staput technique of velocity sedimentation at unit gravity . Populations of 600,000 gonocytes (70-75% purity), sedimenting at about 12 mm/h, could be obtained from 30-35 fetal rats within 8 h after killing . Purities were determined by Nomarski microscopy and verified in fixed preparations and by Coulter volume measurements.

Acta Cytol, 1989 Jul-Aug, 33(4), 544 - 9
Bronchoalveolar lavage fluid processing . Effect of membrane filtration preparation on neutrophil recovery; Thompson AB et al.; Two common methods for the preparation of bronchoalveolar lavage (BAL) fluid for cytologic examination, cytocentrifugation and membrane filtration, have been found to yield different results in the quantitation of lymphocytes . To compare these two methods for the quantitation of neutrophils, the differential counts from 640 consecutive clinical specimens were analyzed retrospectively . The percentage of neutrophils resulting from the preparation of the BAL fluids by the two methods were highly correlated (r2 = .72) . However, cytocentrifugation yielded consistently higher neutrophil percentages than did membrane filtration (means for all samples: 18.5 +/- 1.0% vs . 14.7 +/- 0.9%; P less than .001) . To investigate the source of the variation in neutrophil quantitation by the two methods, two series of mixing experiments were performed in which neutrophil-rich cell suspensions were added to BAL fluids . Determination of the cellular differentials before and after mixing the cell suspensions demonstrated that membrane filtration preparation tends to lose neutrophils while cytocentrifugation accurately recovers neutrophils . Thus, accurate quantitation of the two cells recovered by BAL may require use of both cytocentrifugation and membrane filtration.

Exp Neurol, 1989 Jul, 105(1), 10 - 22
The grafted hippocampus: an epileptic focus; Buzsaki G et al.; Field potentials and unitary activity were investigated in the grafted and the host hippocampi in freely moving rats and in vitro . The subcortical afferents and efferents of the hippocampus (fimbria-fornix, FF) were removed by aspiration . Solid pieces of hippocampal grafts derived from 15- to 16-day-old fetuses were placed in the lesion cavity in rats with unilateral FF lesions, and cell suspensions prepared from fetal hippocampi were grafted directly into the host hippocampi in animals with bilateral FF lesions . Reciprocal communication between the grafted and the host hippocampi was monitored with a 16-microelectrode probe from 7 to 10 months after grafting . The fluorescent retrograde tracer, Fluorogold, was used to examine graft-host projections and acetylcholinesterase staining to reveal host-derived fibers in the graft . The most typical neuronal pattern of the hippocampal graft was a highly synchronous population burst with concurrent EEG spike . The speed of propagation of the EEG spike within the graft and across the graft-host interface was either fast (greater than 3 m/s) or slow (less than 0.5 m/s) . Large amplitude, short duration EEG spikes usually propagated with a high speed, while smaller amplitude, wider spikes with broad population bursts spread at a lower velocity . The direction of propagation was usually uniform indicating that the population burst was triggered by a localized subgroup of highly excitable neurons ("focus") . Spontaneous seizures were also present in the solid graft which frequently invaded the host hippocampus . The incidence of EEG spikes was three times higher in rats with bilateral suspension grafts than in animals with FF lesion only . In about half of the grafted rats spontaneous behavioral seizures were also observed . Intracellular recordings from putative pyramidal cells in the graft and in the host revealed large amplitude (10-12 mV), spontaneously occurring EPSPs . IPSPs were difficult to detect even during depolarizations of up to 20 mV from rest . We suggest that the increased excitability of the hippocampal graft is due to the high incidence of recurrent excitatory collaterals terminating on or close to the somata of pyramidal neurons . Population bursts may spread fast via extensively arborizing axon collaterals or slowly by successively activating new sets of neighboring neurons . Spontaneous behavioral convulsions are explained by assuming that the grafted hippocampus serves as an epileptic focus which is capable of kindling the host brain by repeated seizure induction.(ABSTRACT TRUNCATED AT 400 WORDS)

J Leukoc Biol, 1989 Jul, 46(1), 1 - 10
Development, differentiation, and maturation of fetal mouse yolk sac macrophages in cultures; Naito M et al.; The development and differentiation of macrophages in the fetal mouse yolk sac were studied morphologically in four different culture experiments . In the culture of mouse embryos with yolk sac, the development of fetal macrophages was demonstrated to precede that of promonocytes and monocytes in the yolk sac . In vitro differentiation of the fetal macrophages was consistent with the results of our previous in vivo observation indicating that fetal macrophages were differentiated from primitive macrophages, but not from the monocytic cell series . Differentiation of primitive macrophages into fetal macrophages, before the development of promonocytes and monocytes, was reproduced in the culture of cell suspensions from the fetal mouse yolk sacs, with a mouse bone marrow stromal cell clone (ST2) particularly with those at 8 days of gestation . In the soft agar or liquid culture of yolk sac cells with LP3-conditioned medium, monocyte-macrophage colonies were effectively induced, but not fetal macrophage colonies . The results provide evidence for the existence, in yolk sac hematopoiesis, of two distinct macrophage populations: a fetal macrophage population and a monocyte-derived macrophage population . The data indicate an obvious difference in development and differentiation between the two populations and the temporal precedence of fetal macrophages appearing before monocyte-macrophages.

ASAIO Trans, 1989 Jul-Sep, 35(3), 527 - 30
Extracorporeal hybridization of proximal renal tubular cells and an artificial membrane for the purpose of beta 2 microglobulin removal; Sanaka T et al.; The present study was designed to develop a bioartificial kidney hybridized with LLC-PK1 cells for the purpose of beta 2microglobulin (B2M) removal . Cells were prepared in polystyrene culture wells; the bottom of each was covered with collagen fiber and the cells grown as monolayer cultures . Each well was inoculated with 1 ml cell suspensions with 0.0, 1.0 X 10(6), 2.0 X 10(6), or 4.0 X 10(6) cells/well . B2M was added to the culture system at a concentration of 80 micrograms/dl . Results were as follows: 1) Concentration of B2M in the supernatant of 4 X 10(6) cultivated LLC-PK1 cells was decreased from 79.32 +/- 2.41 micrograms/dl to 67.02 +/- 3.00 micrograms/dl (n = 6, p less than 0.01) at 2 hr; 2) Concentration of B2M in the supernatant of 1 X 10(6), 2 X 10(6), and 4 X 10(6) cultivated LLC-PK1 cells was 75.06 +/- 2.31 micrograms/dl (n = 6, p less than 0.05); 67.27 +/- 11.41 micrograms/dl (n = 6, p less than 0.001); and 67.02 +/- 3.00 micrograms/dl (n = 6, p less than 0.001), respectively, vs . 79.32 +/- 2.41 micrograms/dl in controls at 2 hr; 3) There was a significant positive correlation between the efficacy of B2M removal and cell number; and 4) These results suggested that it is possible to develop an artificial kidney hybridized with proximal tubular cells that possesses a high affinity for B2M removal.

Transplantation, 1989 Jul, 48(1), 87 - 92
Selective proliferation of chemically altered rat liver epithelial cells following hepatic transplantation; Faris RA et al.; Although proliferation of oval cells is often observed during the early stages of chemical hepatocarcinogenesis, the role of these putative hepatic stem cells during the neoplastic process is unknown . In earlier studies our laboratory showed that feeding a choline-deficient (CD) diet containing 0.05% 2-acetylaminofluorene (CD-AAF) to rats produced three subpopulations of oval cells that antigenically resemble biliary duct cells, fetal liver cells, and transitional cells . In the present investigation we have employed a semiallogeneic transplantation protocol in order to study the fate of these nonparenchymal epithelial cells (NPEC) beyond the 4-week endpoint imposed by the lethality of CD-AAF diet . An enriched NPEC suspension containing gamma-glutamyl-transpeptidase (GGT)-positive oval cells (greater than 75%) was isolated from ACI rats maintained on CD-AAF diet for 3 weeks . The donor cells were transplanted via the portal vein into livers of male F1 progeny (LExACI) that had been fed a CD diet for 7 days prior to receiving a partial hepatectomy and the cell suspension . Host rats were then fed either a CD or choline-supplemented (CS) diet for 12 weeks and killed . Colonies of donor-derived cells identified in frozen sections by their lack of reactivity with ACI anti-LE alloantiserum in indirect immunofluorescence (IF) assays were only observed in rats continuously fed the CD diet . Histochemical analysis indicated that the donor-derived colonies expressed GGT, a preneoplastic marker for liver cancer . IF assays using MAbs previously shown to be capable of distinguishing between oval cells and mature hepatocytes indicated that the donor-derived colonies consisted of a mixture of cells with phenotypes resembling those of mature and immature hepatocytes rather than those of oval or ductal cells . Although the cellular origin of the GGT+ donor-derived colonies has not been unequivocally resolved, our results demonstrate that the livers of rats fed a CD-AAF diet contain a chemically altered call population that can be induced to proliferate by a CD diet . In contrast, a CD diet did not promote colonization when normal hepatocytes were employed as the donor cell population, suggesting that the GGT+ oval cells and not the few contaminating GGT- hepatocytes (1%) in the CD-AAF donor cell suspension were the preneoplastic precursors that gave rise to donor-derived colonies . This transplantation protocol will be useful to define the biological potential of chemically altered liver cells during carcinogenesis.

Nippon Ketsueki Gakkai Zasshi, 1989 Jul, 52(4), 767 - 73
Human leukemia cells and mature lymphocytes induce platelet aggregation after removal of cell surface sialic acid; Esumi N et al.; We examined platelet aggregating activity (PAA) of 5 human leukemia cell lines (HL-60, ML-1, HPB-ALL, RPMI-1788, K562), human mature lymphocytes and 2 human neuroblastoma lines (NCG, GOTO) . Although intact cell suspensions of all leukemia cells and mature lymphocytes did not induce platelet aggregation, all cells exhibited PAA in both heparinized and citrated platelet rich plasma (PRP) following neuraminidase treatment (2 units/ml) . In contrast, NCG and GOTO cells with PAA in intact cell suspensions were not affected by neuraminidase . PAA of HL-60 cells pre-cultured in the presence of tunicamycin (0.1-1.0 microgram/ml) to inhibit glycosylation decreased after neuraminidase treatment . Neuraminidase treatment had no effect on procoagulant activity of any of the cells examined . There was no difference in total sialic acid contents between human leukemia and neuroblastoma cells . These results suggest the cell surface glycoconjugates on hematopoietic cells play a role in PAA, and that sialic acid prevents their interaction with platelets.

Cell Tissue Kinet, 1989 Jul, 22(4), 311 - 8
DNA synthesis in Langerhans' cells of mouse ear epithelium revealed by tritiated thymidine autoradiography and histochemical staining for non-specific esterase and beta-glucuronidase activity; Hume WJ et al.; The proportion of Langerhans' cells in DNA synthesis in normal mouse skin was assessed by combining tritiated thymidine {3H}TdR autoradiography with enzyme histochemistry . After injection of {3H}TdR, ear skin was treated in two ways . Epithelial sheet preparations were stained for the presence of non-specific esterase and cytospin preparations of epithelial cell suspensions were stained for beta-glucuronidase activity . The labelling index (+/- SE mean) for cytospins, 40 min after injecting {3H}TdR, was 1.6 +/- 0.15%, doubling to 3-4% from 7-17 days after injection . The sheet preparations showed the proportion of label attributable to paired Langerhans' cells rising from 18% at 40 min after injection, to approximately 45%, on days 1-4 after injection . These results suggest that the proliferation of Langerhans' cells in normal mouse skin might be higher than was previously thought to be the case.

ASAIO Trans, 1989 Jul-Sep, 35(3), 733 - 5
Erythrocyte deformability in patients on left ventricular assist systems; Frattini PL et al.; Preliminary hemorheologic studies using clinical filtration techniques on blood cell suspensions have suggested that changes in erythrocyte (RBC) deformability occur during left ventricular assist system (LVAS) support . In the biophysics literature, it is generally accepted that the elastic properties of the RBC membrane complex affect the microcirculatory deformability of the whole cell (cytoplasmic pathologies excepted) . This paper compares single cell measurements of the surface shear elastic modulus, mu, of the RBC membrane complex (determined using micropipette aspiration) to available clinical filtration pressure data during Novacor LVAS support, over 33 and 126 days in four patients.

J Exp Biol, 1989 Jul, 144, 507 - 20
Metabolism of isolated fish gill cells: contribution of epithelial chloride cells; Perry SF et al.; Gill cell suspensions from freshwater (FW)- and seawater (SW)-adapted teleosts were obtained by density gradient centrifugation . The proportion of chloride cells (CCs) in the mixed cell suspensions was estimated using the fluorescent mitochondrial stain, DASPMEI, and ranged from less than 1% (FW-adapted tilapia) to approximately 13% (SW-adapted toadfish) . The gill cells displayed relatively high viability based on Trypan Blue exclusion (greater than 75%), lactate dehydrogenase leakage (less than 6.5% h-1), oxygen consumption rates (5-15 mumol g-1 cell wet mass h-1) and ATP levels (1-3 mumol g-1 cell wet mass) . There were no obvious differences between the viability of CCs and the other cell types present . An initial comparison of gill oxidative metabolism in SW-adapted tilapia (Oreochromis mossambicus) and toadfish (Opsanus beta) demonstrated that both species oxidized glucose and lactate at substantially greater rates than alanine or oleate . Metabolic rates were significantly higher in toadfish cell suspensions . Kinetic experiments revealed that toadfish gill cells displayed lower values of Km and higher values of Vm for both lactate and glucose, in comparison to tilapia . The elevated metabolism in toadfish gill cells was correlated with increased activities of the oxidative enzyme citrate synthase and Na+/K+-ATPase . The toadfish cell suspensions had a greater proportion of CCs and it is likely that the difference in CC numbers between the two species is the basis for the observed differences in enzyme activities and rates of oxidative metabolism . This idea is supported by the highly significant correlation between Na+/K+-ATPase activity (or CC numbers) and rates of lactate oxidation in gill cell suspensions from FW- and SW-adapted tilapia and toadfish, as well as SW-adapted tilapia chronically treated with cortisol to elevate CC numbers . Although it has been assumed widely that the high metabolic rate of gill tissue reflects, in part, the oxidative demands of the chloride cell, the results of this study provide the first experimental, albeit indirect, evidence for differential rates of metabolism in the various cell types that comprise the gill.

Immunol Invest, 1989 Jul, 18(6), 785 - 95
Multiplication of herpes simplex virus in large granular lymphocytes that co-fractionate with human natural killer cell activity; Strickland P et al.; Highly enriched preparations of human large granular lymphocytes (LGL) cells, isolated from peripheral blood of normal adult donors, showed partial intrinsic resistance to infection with herpes simplex virus type 1 (HSV-1) . Three subsets of LGL cells were identified on the basis of susceptibility to this virus: 1) resistant cells: 2) abortively infected cells; and 3) permissive cells . An average of 25% of LGL cells were completely resistant to infection . The majority (approximately 75%) could be infected as estimated by immunofluorescence . Only 5% of the original cell suspensions were productively infected as determined by infectious center assay and transmission electron microscopy . These results have been reproduced in multiple experiments from 8 different donors consisting of both males and females . No significant difference in LGL cell responses to HSV-1 were detected within this population . Enriched LGL preparations exhibited enhanced natural killer (NK) cell activity . These findings raise several questions concerning the biological significance of LGL susceptibility to infection with HSV-1, relative to virus transport and/or immune surveillance by NK cells.

Br J Haematol, 1989 Jul, 72(3), 445 - 51
On the diagnosis of erythrocyte enzyme defects in the presence of high reticulocyte counts; Lakomek M et al.; The separation of red blood cells into reticulocytes and young and old erythrocytes enables investigations of fractions with different contents of reticulocytes . Activities of hexokinase, glucose phosphate isomerase, phosphofructokinase, pyruvate kinase and glucose-6-phosphate dehydrogenase showed a linear relationship to reticulocyte counts . The dependence of these enzyme activities on the age of the red blood cells exhibited a strong decline from the reticulocyte to the young erythrocyte stage followed by only little further loss of activity, thus leading to a biphasic decay of enzyme activities . By linear regression analysis enzyme activities in erythrocytes (AE) and reticulocytes (AR) could be evaluated . The activity of a given enzyme in the reticulocyte exceeded that of the erythrocyte; the quotient AR/AE represents the decline of enzyme activity from the reticulocyte to the erythrocyte stage . This value AR/AE is 16.7 for pyruvate kinase and 9.4 for hexokinase and thus considerably higher than that for the other enzymes investigated (glucose phosphate isomerase: 2.9, phosphofructokinase: 4.3, glucose-6-phosphate dehydrogenase: 4.5) . In patients suffering from erythrocyte enzymopathies, the AR/AE for pyruvate kinase was 16.2 and thus almost identical to the normal enzyme . Calibration curves where the enzyme activity is plotted versus the fraction of reticulocytes enable the determination of normal activity of a given erythrocyte enzyme depending on the content of reticulocytes in red blood cell suspensions . Thus an unambiguous diagnosis of enzyme defects irrespective of reticulocyte counts becomes possible.

J Neurol Sci, 1989 Jul, 91(3), 323 - 36
Distribution of plasma cells secreting antibodies against nervous tissue antigens during experimental allergic encephalomyelitis enumerated by a nitrocellulose immunospot assay; Zachau AC et al.; The B cell response to central nervous system (CNS) myelin and myelin basic protein, as well as total numbers of IgG secreting cells, was studied in acute experimental allergic encephalomyelitis using a nitrocellulose immunospot assay . The method was able to detect single plasma cells secreting antibodies . Cells secreting antibodies against myelin antigens were detected in regional lymph node cell suspension by day 5 post-immunization (p.i.) . At that time no anti-myelin antibodies were detected free in serum . Later, at day 15 p.i., specific antibody secreting cells were found in bone marrow and spleen indicating a generalization of the immune response . The B cell response became partly sequestered to the target of immune attack since an increased number of IgG secreting cells was detected among mononuclear cells recovered from the CNS . Studies of cellular secretion of antibodies rather than free levels in body fluids may be a more accurate reflection of the in vivo B cell response . These findings may be generally considered in studies of B cell mediated immunity in neuroinflammatory diseases.

Blood, 1989 Jul, 74(1), 156 - 64
Production of colony-stimulating activity by human natural killer cells: analysis of the conditions that influence the release and detection of colony-stimulating activity; Pistoia V et al.; Highly purified natural killer (NK) cell suspensions were tested for their capacity to release colony-stimulating activity (CSA) in vitro . NK cell suspensions comprised primarily CD16+ cells and were devoid of CD3+ T cells, CD15+ monocytes, and of B cells . CSA was detected in the NK cell supernatants and sustained the growth of myeloid colonies from both normal peripheral blood and bone marrow . CSA could be in part inhibited by pretreating NK cell culture supernatants with a specific goat anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antiserum . The inhibition, however, was never complete, a finding that suggests that additional factors were responsible for CSA . Incubation of NK cells with K562 cells (an NK-sensitive target) or with normal bone marrow cells resulted in the appearance of a strong colony-inhibiting activity (CIA) in the culture supernatants . Such CIA was demonstrable in an experimental system where bone marrow or peripheral blood progenitors were induced to form myeloid colonies in the presence of conditioned medium by CSA-producing giant cell tumor (GCT) cells . Stimulation of NK cells with NK-insensitive targets failed to induce CIA production . Neutralizing antitumor necrosis factor (TNF) monoclonal antibodies (MoAbs) were found capable of inhibiting CIA present in the supernatants of NK cells stimulated with K562 cells . Following treatment with anti-TNF antibodies, CSA was again detectable in the same supernatants . This finding indicates that induction of TNF production did not concomitantly switch off CSA production by NK cells . Pretreatment of NK cells with recombinant interleukin-2 (rIL-2) or gamma interferon (r gamma IFN) did not change the amount of CSA released . However, treatment with rIL-2 caused the appearance of a factor in the NK cell supernatants capable of sustaining the formation of colonies of a larger size.

Anticancer Res, 1989 Jul-Aug, 9(4), 1089 - 94
Cytotoxic drug sensitivity of squamous cell carcinoma as predicted by an in vitro testing model; Elprana D et al.; An in vitro technique for testing the chemosensitivity of squamous cell carcinoma from the head and neck is described . Single-cell suspensions and tumor slices obtained from human xenografts were used . The sensitivity to cisplatin and bleomycin was quantified using the incorporation of (3H) thymidine as a parameter . The in vitro assays were compared with in vivo tests in mice . No reliable data could be obtained with cell suspensions of these tumors due to a rapid decrease in cell viability during incubation, while tumor slices revealed a stable control level of (3H) thymidine incorporation for up to 30 hours . In 6 out of 7 cases close agreement was found between the effect of the cytotoxic drug on the tissue slices and on the same tumors in nude mice.

Acta Cytol, 1989 Jul-Aug, 33(4), 550 - 6
Simultaneous enumeration of T-cell subsets and macrophages in bronchoalveolar lavage fluids by immunoenzyme double staining . Comparison with conventional immunofluorescence; van Maarsseveen TC et al.; Bronchoalveolar lavage is a common research and clinical tool for the retrieval of cells from the lower respiratory tract . In addition to conventional morphologic study of these cells, the subtyping of T lymphocytes is often important for reaching a diagnosis of a disease or assessing its activity; subtyping is usually done by a standard immunofluorescence assay on cell suspensions requiring about 5 X 10(5) cells . Since the number of leukocytes in the lavage fluid from many patients is too small to obtain reliable information by this assay, a double immunoenzyme staining of T-lymphocyte subtypes on Cytospin preparations was utilized . This method, which requires a small number of cells, was compared with the standard immunofluorescence assay for the identification and quantitation of lymphocyte subtypes in the lavage fluids of patients with different disorders . Although the immunoenzyme double staining assay is somewhat more laborious, it provides important advantages: (1) simultaneous observation of two lymphocyte subsets and macrophages on the same slide; (2) a considerably smaller number of cells (2 X 10(4) instead of 5 X 10(5} is necessary; (3) the availability of permanent preparations; (4) the possibility of storing the Cytospin slides before staining; and (5) conventional light microscopy can be used . Since the reliability of both techniques appeared to be the same, the double staining assay for routine usage with bronchoalveolar lavage fluids appears to be preferable.

Lab Invest, 1989 Jul, 61(1), 98 - 106
New monoclonal antibody that specifically recognizes murine interdigitating and Langerhans cells; Maruyama T et al.; A new monoclonal antibody, M1-8, that recognizes murine interdigitating cells (IDC) and Langerhans cells was obtained from a hybridoma prepared by fusion of SP2/0 mouse myeloma cells with splenic cells of rats immunized with IDC-rich cell suspension obtained from lymph nodes of athymic nude mice (BALB/c nu/nu) . The specificity was assessed immunohistochemically on frozen sections of lymph nodes and epidermal sheets from both nude and normal mice . M1-8 reacted with paracortical IDC, veiled cells of the marginal sinus, and epidermal Langerhans cells in both normal and nude mice . In simultaneous staining by M1-8 and nonspecific esterase or anti-Ia or anti-Thy-1,2 antibody, the same epidermal dendritic cells were positive for all these antigens except Thy-1,2 . Immunoelectron microscopy of the lymph node suspension using gold colloid particles revealed the attachment of gold particles to the cell membrane of IDC . Analysis by flow cytometry of the lymph node cell suspension showed 14 or 6% of M1-8-positive cells in nude or normal mouse, respectively . Immunohistochemical analysis showed that M1-8 also reacted with dendritic cells in the thymus and spleen and had a different distribution from F4/80 . M1-8 also reacted with monocytes in bone marrow and peripheral blood, alveolar macrophages, and thioglycollate-stimulated peritoneal exudate macrophages . The antibody belongs to the immunoglobulin M class, reacts immunochemically with a glycoprotein in the cell membrane, and has a molecular mass of approximately 15 kDa.

Nippon Gan Chiryo Gakkai Shi, 1989 Jun 20, 24(6), 1261 - 5
{The effect of a static magnetic field on hamsters bearing melanoma--cell cycle analysis by flow cytometry}; Imajo Y et al.; Hamsters bearing Greene's melanoma in the side were exposed to an almost uniform 0.2 Tesla (T) magnetic field for three hours . Tumors were extirpated immediately after and 3, 12 and 21 hours after the exposure, then were minced and meshed into a single cell suspension . Cells were stained with anti BrdU FITC and Propidium Iodide for cell cycle analysis with FACStar . The distribution of each cycle phase of the exposed group was somewhat unequal to that of the control group . Namely, there was a decrement of G1% at three and twelve hours after exposure and an increment of S% (p = 0.01) at twelve hours after exposure.

Ned Tijdschr Geneeskd, 1989 Jun 10, 133(23), 1179 - 83
{Peroperative transfusion of autologous blood; experiences with a blood cell separator}; Koopman-van Gemert AW; Technical progress in the last decade has made intraoperative autotransfusion of shed blood by means of a blood cell separator a safe method of blood transfusion . Complications may now be prevented by removing potentially harmful substances such as debris from the wound, heparin, activated clotting factors and free haemoglobin . The Red Cross Blood Bank in Nijmegen has performed 80 intraoperative autotransfusions . An average of 4 litres of blood was processed and 1.2 litres of red cell suspension were prepared, with a haematocrit of 0.47 l/l . This means saving about 3 donor units of packed cells during operation . Recovery of the erythrocytes was 61.9%; of the heparin and haemoglobin 95.3 and 95%, respectively, were removed . Almost all platelets were removed . No complications were noted . This technique can reduce intraoperative blood loss and use of donor blood.

Ned Tijdschr Geneeskd, 1989 Jun 10, 133(23), 1174 - 8
{Experiences in clinic and blood bank with a program for preoperative donation of autologous blood}; van de Wiel A et al.; In the period from March 1987 to January 1988 a programme for donation of autologous blood was carried out within the existing cooperation between a regional blood bank and a general hospital . Various organizational problems were avoided by means of a special consulting hour and introduction of a special consultation form . Blood was taken a total of 144 times from 49 orthopaedic patients . No serious problems were encountered; three times, withdrawal of a unit of blood had to be refrained from . Of the 144 units of red cell suspension, 107 (73.3%) were reinfused, while seven patients still required administration of homologous blood . The variations of haemoglobin level, haematocrit, reticulocyte count and red cell distribution width suggest stimulation of erythropoiesis by repeated withdrawal of blood at brief intervals . In the preoperative phase, there were no pronounced differences in this respect between patients who were and those who were not given supplementary iron . Autologous blood transfusion constitutes a safe, valuable addition to the current transfusion forms and is possible within the existing organizations in The Netherlands.

Neurosci Lett, 1989 Jun 5, 101(1), 17 - 22
Recovery of hippocampal cholinergic activity by transplantation of septal neurons in AF64A treated rats; Ikegami S et al.; Embryonic septal neurons were transplanted into the hippocampus of adult rats which had received lateral-ventricular administration of AF64A, a cholinergic neurotoxin, and the effects on hippocampal cholinergic activity were studied . One week after AF64A administration, we injected dissociated septal cell suspension into the dorsal hippocampus, unilaterally . About 3 months after the transplantation, acetylcholine (ACh)-rich septal grafts formed extensive acetylcholinesterase (AChE)-positive fibers into the host hippocampus, recovering choline acetyltransferase (ChAT) level only in the grafted side . These results indicate that septal implants can produce a partial recovery of the cholinergic activity in the chemically damaged hippocampus.

J Immunol Methods, 1989 Jun 2, 120(1), 23 - 7
Measurement of cytoplasmic calcium in lymphocytes using flow cytometry . Kinetic studies and single cell analysis; Griffioen AW et al.; An increased level of cytoplasmic free ionized calcium {Ca2+}i after crosslinking of membrane receptors is a critical second messenger in the activation of T and B lymphocytes . The availability of fluorescent calcium chelators, such as quin-2 and indo-1, makes accurate measurement of {Ca2+}i possible . One of the major drawbacks of spectrofluorometry which is the generally used method in such studies is that the overall response of a cell suspension is recorded . Such data will be biased by the proportion of non-responding cells, which will differ according to the purity of cell populations and the nature of the stimulus applied . An accurate and reliable technique to measure intracellular free calcium responses in indo-1-loaded cells at the single cell level has been developed using a simple mercury arc lamp-based flow cytometer, the FACS analyzer . Using this technique we have found that the rapid increase in {Ca2+}i (within 30 s) in T cells following activation by ConA involves a minority of cells, whereas all T cells show increased {Ca2+}i levels within 2-3 min.

Histochem J, 1989 Jun, 21(6), 348 - 56
Localization of molecules with restricted patterns of expression in morphogenesis: an immunohistochemical approach; Thibodeau A et al.; In a search for molecules with restricted patterns of expression during development, monoclonal antibodies were raised against different transitory structures of the chick embryo . Mice were immunized with cell suspensions from lightly homogenized embryonic tissues explanted from morphogenetically active regions . A convenient immunohistochemical assay was used to screen the hybridoma supernatants on a large scale . It relied on the use of poly(ethylene glycol) as embedding medium . Its water miscibility allowed, in a one-step incubation with antibody-containing supernatants, the dewaxing and rehydration of the tissue sections as well as antibody binding . We report here the usefulness of this approach in selecting monoclonals with unique patterns of immunoreactivity . In this study, cephalic neural crest cells in early or late phase of migration, together with their surrounding tissues, were used as immunogens . The monoclonal antibodies obtained have been classified into regional, cell-lineage, cell-cycle or extracellular material-associated markers . The information provided by the direct visualization of the immunoreactivity of the various monoclonal antibodies on tissue sections, as early as the first round of screening, allows rapid determination of the subsequent strategy to be followed for further characterization of the individual markers.

J Invest Dermatol, 1989 Jun, 92(6), 842 - 7
Decreased number and function of antigen-presenting cells in the skin following application of irritant agents: relevance for skin cancer?
Lisby S, Baadsgaard O, Cooper KD, Vejlsgaard GL.
The mechanism of irritant dermatitis and the immunologic consequences of such reactions are unclear . We evaluated the number and function of epidermal antigen-presenting cells contained in epidermal cell suspensions obtained from normal and irritant patch test reaction sites . Application of sodium lauryl sulfate or croton oil to human skin in vivo resulted in a progressive depletion in the number of epidermal OKT6+HLA-DR+ (T6+DR+) Langerhans cells (LC) from 3.1 +/- 0.2% of total epidermal cells (EC) to 1.2 +/- 0.1% after 8 d (mean values +/- SEM, N = 9) . Between 1-4 d irritant patch test sites demonstrated an influx of non-Langerhans cell T6-DR+ cells . These cells were not DR+ keratinocytes but appeared to be of bone marrow derivation because they expressed the marker, HLe1 . Among bone marrow derived cells, the T6-DR+EC appeared to be of monocyte, macrophage lineage, because they expressed the determinant recognized by the OKM5 (M5) antibody . Despite the induction of M5+DR+EC the total number of DR+EC showed progressively decreasing percentages over an 8-d period . Partial recovery to 73 +/- 12% of control value was observed at 2 weeks, with full recovery by 4 weeks after challenge . Concomitantly with the depletion of DR+EC, the capacity of EC to present alloantigens to T cells decreased . This reduction in antigen-presenting cell activity was strongly correlated to the reduction in total DR+ EC (r = 0.94, p less than 0.05) . Thus, the capacity of irritants such as croton oil to abrogate the function of epidermal antigen-presenting cells may be related to the tumor promoting potential of these agents.

Arch Otolaryngol Head Neck Surg, 1989 Jun, 115(6), 725 - 30
Production of lymphokine-activated lymphocytes . Lysis of human head and neck squamous cell carcinoma cell lines; Alessi DM et al.; Lymphokine-activated killer cells are thought to be important mediators of host tumor defense . In the present study, the cytotoxic potential of lymphokine-activated lymphocytes against different head and neck squamous cell carcinoma cell lines was investigated . Lymphokine-activated killer cells were derived from peripheral blood lymphocytes . Effector peripheral blood lymphocyte cell suspensions were incubated in the presence or absence of recombinant interleukin-2 . Cytotoxicity of incubated cells or fresh peripheral blood lymphocytes was determined in a 3-hour chromium 51 release assay . Target cell lines included K562 (a natural killer-sensitive target) and the following head and neck squamous cell carcinoma cell lines: Cal 27, UMSCC-1, UMSCC-8, UMSCC-16, UMSCC-19, and UMSCC-22a . Fresh peripheral blood lymphocytes and peripheral blood lymphocytes cultured in the absence of added interleukin-2 demonstrated minimal cytotoxic effects against the squamous cell carcinoma targets . In contrast, these fresh and incubated lymphocytes showed significant cytotoxic effects against K562 . Cells preincubated in the presence of interleukin-2 demonstrated a statistically significant increase in cytotoxic effects against K562 and all squamous cell carcinoma targets . These investigations support the possible role of lymphokine-activated killer cells in host defense against squamous cell carcinoma . In vitro natural killer cell activity against head and neck squamous cell carcinoma cell lines is low; however, significant lymphokine-activated killer cell cytotoxicity is present.

J Neurosci Res, 1989 Jun, 23(2), 198 - 206
Purification and culture of adult rat dorsal root ganglia neurons; Delree P et al.; To study the trophic requirements of adult rat dorsal root ganglia neurons (DRG) in vitro, we developed a purification procedure that yields highly enriched neuronal cultures . Forty to fifty ganglia are dissected from the spinal column of an adult rat . After enzymatic and mechanical dissociation of the ganglia, myelin debris are eliminated by centrifugation on a Percoll gradient . The resulting cell suspension is layered onto a nylon mesh with a pore size of 10 microns . Most of the neurons, the diameter of which ranged from 17 microns to greater than 100 microns, are retained on the upper surface of the sieve; most of the non-neuronal cells with a caliber of less than 10 microns after trypsinization go through it . Recovery of neurons is achieved by reversing the mesh onto a Petri dish containing culture medium . Neurons to non-neurons ratio is 1 to 10 in the initial cell suspension and 1 to 1 after separation . When these purified neurons are seeded at a density of 3,000 neurons/cm2 in 6 mm polyornithine-laminin (PORN-LAM) coated wells, neuronal survival (assessed by the ability to extend neurites), measured after 48 hr of culture, is very low (from 0 to 16%) . Addition of nerve growth factor (NGF) does not improve neuronal survival . However, when neurons are cultured in the presence of medium conditioned (CM) by astrocytes or Schwann cells, 60-80% of the seeded, dye-excluding neurons survive . So, purified adult DRG neurons require for their short-term survival and regeneration in culture, a trophic support that is present in conditioned medium from PNS or CNS glia.(ABSTRACT TRUNCATED AT 250 WORDS)

Gen Comp Endocrinol, 1989 Jun, 74(3), 400 - 5
Enhanced progesterone and testosterone secretion and depressed estradiol secretion in vitro from small white follicle cells of incubating turkey hens; Porter TE et al.; This study was conducted to determine if lower steroid secretion by the small white follicles in incubating turkey hens contributes to lower circulating steroid concentrations during this time . Turkey hens were grouped as either laying or incubating . Serum samples and the ovarian small white follicles (SWF; 2-7 mm diameter) were collected from each hen . The SWF were pooled for each group and their cells were dispersed by trypsin digestion . Serum-luteinizing hormone (LH), progesterone (P), testosterone (T), and estradiol (E) concentrations were lower and serum prolactin concentration was higher during incubation than during egg laying . SWF cells from incubating hens secreted more P and T and less E in response to ovine luteinizing hormone (oLH) than did similar cell suspensions from laying hens . The incubating hens' SWF cells' capacity to secrete E but not their capacity to secrete P or T in vitro is consistent with the observed circulating levels . It is hypothesized that lower levels of circulating LH and/or higher levels of prolactin found in incubating hens may have a depressing effect on aromatase activity and/or an up-regulating effect on LH-induced P and T secretion by the SWF cells.

J Urol, 1989 Jun, 141(6), 1483 - 7
Characterization and localization of in vivo phospholipid methylation in the hamster testis; Schlegel PN et al.; Although previous studies have demonstrated that phospholipid methylation occurs in the testis and may be involved in Leydig cell function, phospholipid methylation in spermatogenic cells has not been characterized . In this study we describe the occurrence, time course, and localization of phospholipid methylation in the hamster testis following intratesticular injection of radioactive methyl precursor . Adult and pubertal (seven day old) hamsters were injected intratesticularly with {3H-methyl}-methionine and sacrificed 10 min . to 31 hours thereafter . The testes were then removed and homogenized or dispersed into cell suspensions . Spermatogenic cell and Leydig cell enriched preparations were isolated from the dispersed cell preparations using elutriation and Percoll gradient centrifugation and assayed for methylated phospholipids and proteins . These experiments demonstrated that 1) phospholipid methylation occurs in the hamster testis at a level seven-fold greater than protein methylation, 2) the incorporation of radioactivity associated with phospholipid methylation is progressive over time, and 3) in vivo, spermatogenic cell preparations enriched with pachytene spermatocytes have an almost four-fold higher level of measurable phospholipid methylation when compared to whole testis preparations . Taken together, these results suggest that phospholipid methylation may play an important stage-specific role in spermatogenesis.

J Neurochem, 1989 Jun, 52(6), 1741 - 50
Determination of brain interstitial concentrations by microdialysis; Benveniste H et al.; Microdialysis is an extensively used technique for the study of solutes in brain interstitial space . The method is based on collection of substances by diffusion across a dialysis membrane positioned in the brain . The outflow concentration reflects the interstitial concentration of the substance of interest, but the relationship between these two entities is at present unclear . So far, most evaluations have been based solely on calibrations in saline . This procedure is misleading, because the ease by which molecules in saline diffuse into the probe is different from that of tissue . We describe here a mathematical analysis of mass transport into the dialysis probe in tissue based on diffusion equations in complex media . The main finding is that diffusion characteristics of a given substance have to be included in the formula . These include the tortuosity factor (lambda) and the extracellular volume fraction (alpha) . We have substantiated this by studies in a well-defined complex medium (red blood cell suspensions) as well as in brain . We conclude that the traditional calculation procedure results in interstitial concentrations that are too low by a factor of lambda 2/alpha for a given compound.

Burns, 1989 Jun, 15(3), 155 - 61
Thymic response to thermal injury in mice: I . Alterations of thymocyte subsets studied by flow cytometry and immunohistochemistry; Colic M et al.; The dynamics of thymocyte subset changes in mice subjected to sublethal thermal injury were studied in cell suspensions by flow cytometry and in situ by immunohistochemistry . Thermal injury caused acute thymic involution in the first 2 days which was the consequence of a considerable decrease in numbers of Thyl.2high+ CD4+ CD8+, cortical thymocytes . Medullary, Thyl.2low+ thymocytes were more resistant and their relative values increased . In the regenerative phase (2-14 days) the recovery of large CD4- CD8-, early thymocytes, mainly localized in the subcapsular area of the thymus, preceded the regeneration thymocytes of the cortical phenotype . Judged by the absolute numbers of medullary thymocytes it can be seen that CD4+ CD8- (T-helper/inducer cells) were more sensitive to the effect of thermal injury than CD4- CD8+ (T-suppressor/cytotoxic cells) . While values of CD4+ CD8- cells were constantly and progressively lower during 2 weeks after thermal injury, absolute numbers of CD4- CD8+ cells showed cyclic changes with lower and higher values compared to controls . An increase in the numbers of CD4- CD8+ cells was found at day 6 after thermal injury.

J Med Microbiol, 1989 Jun, 29(2), 101 - 10
Silver resistance in Escherichia coli R1; Starodub ME et al.; Escherichia coli strain R1, originally isolated from a patient whose burns were treated with silver sulphadiazine, contained two large plasmids of 83 kb (pJT1) and 77 kb (pJT2), and was resistant to 1 mM AgNO3 . A silver-sensitive derivative, E . coli S1, cured of the 83-kb plasmid pJT1, was obtained by growth at 46 degrees C . Studies with an Ag+-specific ion electrode showed no significant differences in Ag+ binding by washed resting cell suspensions of strains R1 and S1, with and without glucose . However, transmission electronmicroscopy and energy dispersive X-ray analysis of whole cell mounts from actively growing cultures showed that the Ag+-resistant strain did not accumulate Ag+, whereas the sensitive strain contained dense silver particles . Both strains produced H2S, detected by blackening of lead acetate paper above inoculated broth, and reducing substances (possibly H2S) were detected only around E . coli R1 colonies when methylene blue was used as a indicator in LB agar, which may be a less sensitive assay . The mechanism of silver resistance is not known, but actively growing cells of E . coli R1 did not accumulate silver.

Scand J Clin Lab Invest, 1989 Jun, 49(4), 323 - 7
Effect of freezing and ultrasonication on the density of human lymphocyte beta 2-adrenoceptors; Santala M et al.; In this study 21 pregnant volunteers were divided into three equal groups; blood samples were taken from each, and after isolation of the lymphocytes the cell suspensions were divided into two equal samples . The density of beta 2-adrenoceptor in intact lymphocytes was compared with the receptor density for lymphocytes stored 7 days or 19 days at -70 degrees C and with cells disrupted by ultrasonication . Storage by lymphocytes at -70 degrees C for 7 days to 19 days decreased the beta 2-adrenoceptor density by 20% and 37%, respectively; ultrasonication decreased the density by 65%.

Exp Cell Res, 1989 Jun, 182(2), 645 - 52
Immunocytochemical distribution of extracellular matrix receptors in human osteoclasts: a beta 3 integrin is colocalized with vinculin and talin in the podosomes of osteoclastoma giant cells; Zambonin-Zallone A et al.; Human osteoclasts (OCLs) obtained from cell suspensions of surgically excised giant cell bone tumors (osteoclastomas) were attached to glass coverslips and analyzed by immunofluorescence with antibodies to integrins and cytoskeletal proteins . It was found that in OCLs (i) podosomes, identified by their F-actin core and by interference reflection microscopy, were predominantly found in a peripheral belt as described in avian OCLs; (ii) each F-actin core was surrounded by a ring of vinculin and talin; (iii) beta 1 integrin was diffuse in the ventral membrane; (iv) beta 3 integrin was distributed in intensely fluorescent rings surrounding F-actin cores; (v) beta 2 integrin was absent; (vi) beta 4 integrin was absent . The macrophages detected in the same coverslips displayed podosomes containing beta 2 but not beta 3, fibroblasts showed adhesion plaques positive for beta 1 and beta 3 but not for beta 2, and platelets were intensely positive for beta 3 . These results indicate that OCLs produce an integrin complex that is absent in the monocyte-macrophage lineage.

Endocrinology, 1989 Jun, 124(6), 2995 - 3002
Antagonism of contractants and relaxants at the level of intracellular calcium and phosphoinositide turnover in the rat uterus; Anwer K et al.; The effects of the uterine relaxants relaxin and isoproterenol on intracellular free calcium and inositol phosphate formation were investigated in rat myometrium . Preincubation of fura-2-loaded myometrial cell suspensions with relaxin and isoproterenol inhibited the oxytocin-induced stimulation of intracellular free calcium, with EC50 values of 0.02 and 1.0 microM, respectively . Pretreatment of cells with pertussis toxin or replacement of extracellular calcium with 2 mM EGTA inhibited the oxytocin-induced increase in intracellular calcium by 47% and 50%, respectively, but did not inhibit the action of relaxin . (Bu)2cAMP and forskolin also inhibited the effect of oxytocin on intracellular calcium . In uterine strips prelabeled with {3H}inositol, oxytocin stimulated a dose-dependent accumulation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate, with and EC50 of 0.38 microM, and pertussis toxin inhibited this effect . Relaxin, isoproterenol, chlorophenylthio-cAMP, and forskolin inhibited the oxytocin-stimulated formation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate . The effect of relaxin on inositol trisphosphate formation was dose dependent, with an EC50 of 0.1 microM . Relaxin and isoproterenol also inhibited inositol phosphate formation in myometrial cells . These data demonstrate the attenuation of contractant-induced elevation in myometrial intracellular calcium and phosphoinositide turnover by uterine relaxants and suggest that these actions may be related . In addition, they provide additional evidence that cAMP-mediated mechanisms may be involved in mediating uterine relaxation.

Gastroenterology, 1989 Jun, 96(6), 1434 - 8
Parietal cell antibodies in pernicious anemia inhibit H+, K+-adenosine triphosphatase, the proton pump of the stomach; Burman P et al.; Antibodies to a membrane-bound antigen, localized to the canalicular structures of the parietal cell, are found in most sera of patients with chronic atrophic gastritis and pernicious anemia . In the present study immunoglobulins containing parietal cell antibodies were found to inhibit the activity of H+,K+-adenosine triphosphatase (EC 3.6.1.36) in a tubulovesicular membrane preparation from porcine gastric mucosa . The degree of inhibition correlated to the titer of parietal cell antibodies as assessed by an enzyme-linked immunosorbent assay . The specificity of the enzymatic inhibition was confirmed by the lack of effect of parietal cell antibodies on membrane-bound esterase . A possible interaction of parietal cell antibodies with gastrin binding at the receptor level was investigated in a radioreceptor assay employing 125I-gastrin 1 and gastric mucosal cell suspension from the guinea pig . No blocking capacity was found with immunoglobulins from patients with pernicious anemia as compared with immunoglobulins from healthy controls . The results thus demonstrate a direct inhibitory effect of parietal cell antibodies on the acid producing H+,K+-adenosine triphosphatase of the parietal cell, but also a lack of interaction with the gastrin receptor, and indicate that in the development of hypo/achylia H+,K+-adenosine triphosphatase autoantibodies could have a major pathogenic role.

Eur J Immunol, 1989 Jun, 19(6), 1087 - 93
Proliferation of lymphocyte subsets in the adult rat: a comparison of different lymphoid organs; Westermann J et al.; Adult, male Lewis rats received a single injection of 5-bromo-2'-deoxyuridine (BrdUrd) i.v . to label proliferating cells in the S phase of the cell cycle . After 1 and 24 h the thymus, bone marrow, blood, spleen, peripheral, cervical and mesenteric lymph nodes as well as Peyer's patches were removed . In cell suspensions surface staining was performed for B, T, T helper (Th) and cytotoxic/suppressor (Tc/s) T lymphocytes by identifying kappa light chain, CD5+, CD4+ and CD8+ cells, respectively . On the same slide the DNA label BrdUrd was demonstrated by a monoclonal antibody . B, T, Th and Tc/s lymphocytes proliferate locally both in central lymphoid organs such as the thymus and the bone marrow, and in peripheral lymphoid organs such as the spleen, lymph nodes and Peyer's patches . Within an organ the amount of proliferation among the lymphocyte subsets is similar, differing not more than threefold . Although concerning only a small fraction of cells within the organ, an unexpected finding is the high percentage of BrdUrd-labeled cells among B lymphocytes in the thymus (3%) and among T lymphocytes in the bone marrow (3%) . One day after injection of BrdUrd the thymus contains 25% BrdUrd+ T lymphocytes, while the other organs investigated do not show more than about 2% BrdUrd+ B and T lymphocytes . Many of the newly formed lymphocyte subsets leave their organ of birth within 24 h . Thus the amount of proliferation in the lymphocyte subsets investigated is very similar and the differences between central (thymus and bone marrow) and peripheral lymphoid organs are much smaller than expected.

Scand J Immunol, 1989 Jun, 29(6), 671 - 7
Effects of purified protein derivative (PPD)-activated syngeneic epidermal cells on a PPD-specific rat T-helper cell line; Scheynius A et al.; An important question in local immune regulation in the skin is how keratinocytes at inflammatory sites can modify a local T-cell response to antigens introduced via the skin . In the present study we investigated the effects of rat epidermal cells obtained from the site of a tuberculin reaction, on the proliferative response of a syngeneic purified protein derivative (PPD)-specific CD4+ T-cell line . Epidermal cell suspensions from the tuberculin-reactive ears contained 23-37% cells expressing class II transplantation antigens as judged by immunocytochemistry compared with 2-3% in normal epidermis . When comparing the capacity of these two different epidermal cell populations to induce a PPD-specific T-cell response in vitro, it was found that the PPD-reactive epidermal cells induced a lower T-cell response than did normal epidermal cells . This discrepancy cannot be explained by an infiltration of inflammatory cells into the epidermis of tuberculin-reactive ears . Our data indicate that epidermal cells modified during a delayed-type hypersensitivity reaction in vivo may suppress an antigen-specific T-cell proliferation.

Arch Biochem Biophys, 1989 Jun, 271(2), 488 - 94
S-adenosyl-L-methionine:trans-caffeoyl-coenzyme A 3-O-methyltransferase from elicitor-treated parsley cell suspension cultures; Pakusch AE et al.; An S-adenosyl-L-methionine:caffeoyl-CoA 3-O-methyltransferase was purified 82-fold from elicitor-induced parsley cell suspension cultures by ammonium sulfate fractionation, anionic exchange and hydrophobic interaction chromatographies, and chromatofocusing . The enzyme has an apparent pI of 5.7 and a molecular weight of approx 48,000 determined by gel filtration chromatography . Maximal activity was observed at pH 7.5 in 50 mM phosphate or Tris-HCl buffers and the additional presence of 0.5 M NaCl . The methyltransferase activity was dependent on Mg2+, whereas EDTA, Mn2+, and Ca2+ inhibited the reaction . The partially purified enzyme efficiently catalyzed the methylation of caffeoyl-CoA, but also accepted with low affinity various other caffeic esters as substrates . Dark-grown parsley cells contained considerable methyltransferase activity which was nevertheless increased approx threefold within 12 h following the addition of a crude fungal elicitor to the cell suspensions . We propose that the O-methyltransferase activity is an important component in the rapid resistance response of the cells, which depends on the formation of cell wall-bound ferulic polymers.

Tsitologiia, 1989 Jun, 31(6), 690 - 5
{DNA degradation and changes in the permeability and form of Ehrlich ascites tumor cells when incubated in an anaerobic medium without glucose}; Proskurikov SIa et al.; After a 3-hour incubation of the Ehrlich ascite tumor cells in buffered Hanks solution, without glucose and oxygen, the extensive cell injuries were observed . The time-course of appearance of these injuries was as follows: cell blebbing, staining of the cells with trypan blue, and then their staining with ethidium bromide . The DNA degradation registered with fluorometric method coincided in time with cell staining with trypan blue . All injuries (except DNA degradation) were delayed at pH 6.0 compared with those at pH 7.3 . Glucose added to the cell suspension greatly protected the cells from these injuries, although DNA degradation at pH 6.0 in these conditions was a little higher than that at pH 7.3.

J Chromatogr, 1989 May 24, 470(1), 251 - 60
High-voltage capillary zone electrophoresis of red blood cells; Zhu A et al.; The high-voltage wide-bore capillary zone electrophoresis of red blood cells was investigated . The reproducibility of the retention time (electrophoretic mobility) is excellent and the differentiation among various species is good . The peaks in the electropherogram describe the distribution of the size and/or surface charge of the cells and are therefore broad . The relationship between the peak height and the number of cells injected is good, with linear correlation coefficients better than 0.98 . Details of the preparation of cell suspensions and support electrolytes are given, which is essential for obtaining reproducible results . The inner surface of FEP capillary tubing is degraded by the application of high voltage and a pause is necessary between successive experiments if good and reproducible peak shapes are to be obtained . The length of the pause increases with the number of experiments made, and finally the tubing becomes useless . Inspection of the inner surface by X-ray photoelectron spectroscopy showed the breakdown of CHF bonds, but the actual mechanism is not known.

Biochem Int, 1989 May, 18(5), 981 - 90
Possible generation of hydrogen peroxide and lipid peroxidation of erythrocyte membrane by asbestos: cytotoxic mechanism of asbestos; Iguchi H et al.; We studied a mechanism of hemolysis induced by asbestos particles or silicic acid . This hemolysis was instantly initiated by mixing red blood cells with asbestos particles or silicic acid, and reached a plateau within 10 min . The hemolysis was suppressed by catalase, radical quenchers, deoxygenation, or phospholipids . The degree of the hemolysis was proportional to either the amount of asbestos added into red blood cell suspension or the amount of thiobarbituric acid-reacting substances formed . These findings suggest that in vitro hemolysis induced by asbestos particles (or silicic acid) is ascribed to membrane lipid peroxidation initiated by hydrogen peroxide which was generated by the interaction of the mineral particles with biological membranes.

Arch Exp Veterinarmed, 1989 May, 43(3), 455 - 61
{A simple method for the quantitative determination of sperm aggregation}; Blottner S et al.; Described in this paper are 2 methodological variants for photometric recording of sperm aggregation . Heparin was used to induce aggregation . One of the working principles was related to aggregation-associated alteration of turbidity in an agitated cell suspension, measured by the KZM-1 coagulation-time meter, in analogy to measurement of thrombocyte aggregation . The 2nd variant was based on variation of cloudiness due to sedimentation of aggregates in a non-agitated suspension . Both methods provided equally valid information . They characterised both intensity of aggregation as a combined effect of the number and size of aggregates as well as the rate of aggregation . Photometric recording has proved to be an objectivated method for quantitative assessment of aggregation . Its use is proposed for studies into capacitation or immunological response of spermatozoa.

Zentralbl Veterinarmed B, 1989 May, 36(3), 191 - 8
Granulocyte isolation from whole blood of goat, sheep, cattle, horse, dog, pig, and man; Reifenberg K et al.; A simple two step procedure for the isolation of caprine, ovine, bovine, equine, canine, porcine and human peripheral blood granulocytes is described . After enrichment of granulocytes by centrifugation, contaminating erythrocytes are lysed hypotonically . Recovery, purity, and viability of the granulocyte suspensions are determined . FACScan analysis of the cell suspensions measuring cellular size by forward and sideward light scatter is compared with the corresponding analysis of whole blood leukocytes . Constituencies of the isolated cell suspensions and loss of granulocyte subpopulations through isolation procedure is discussed with regard to granulocyte function assays.

Nippon Sanka Fujinka Gakkai Zasshi, 1989 May, 41(5), 557 - 63
{Morphological and functional changes in 3 beta-HSD positive cells in corpora lutea during pregnancy in rats}; Miyauchi F et al.; On days 5, 10, 15 and 20 of pregnancy, rat corpora lutea (CLs) were dissected and dissociated into single cell suspensions by enzyme treatments . To assess 3 beta-hydroxysteroid dehydrogenase (HSD) activity, a histochemical suspension-staining procedure was used . The number of cells positive for 3 beta-HSD in CL were 148.0 +/- 13.7, 130.1 +/- 25.4, 134.0 +/- 23.5 and 116.8 +/- 13.5 X 10(3) cells on days 5, 10, 15 and 20 of pregnancy, respectively . The 3 beta-HSD positive cells increased in size from 18.5 +/- 0.28 microns on day 5 to 35.7 +/- 0.50 microns on day 20 of pregnancy . The suspended luteal cells were incubated in serum-free DME-F12 for 20 hours with or without human chorionic gonadotropin (hCG, 100 ng/ml) or dibutyryl cyclic AMP (dbcAMP, 10mM) to test their functionality, and progesterone accumulation was determined by radioimmunoassay . Progesterone secretion from the 3 beta-HSD positive cells was maintained at the same levels until day 15 and decreased significantly on day 20 of pregnancy . However the response to hCG stimulation of the 3 beta-HSD positive cells decreased significantly on day 20, the 3 beta-HSD positive cells maintained the same responsiveness to dbcAMP stimulation on progesterone secretion throughout pregnancy . These data suggest that the steroidogenic rat luteal cells may be regulated by morphologically and functionally different mechanisms, and that progesterone may be secreted by at least two different pathways.

Respir Physiol, 1989 May, 76(2), 191 - 203
Analysis of oxygen binding by Xenopus laevis hemoglobin: implications for the Root effect; Kister J et al.; We have measured oxygen binding properties for red cell suspensions and stripped hemolysate of Xenopus laevis (XL) in order to answer the question whether XL hemoglobin exhibits a Root effect . The present results show that under physiological conditions XL red cells do not exhibit all the criteria for a Root effect . Compared to human Hb A, stripped XL hemoglobin has a low oxygen affinity, a normal alkaline Bohr effect and a lower interaction with 2,3-diphosphoglycerate, its physiological allosteric effector in red cells . The values for the Hill coefficients are lower than those for human hemoglobin but XL Hb remains cooperative (n50 approximately 2), even at pH values below 6 . Attempts to mimick some of the criteria for the Root effect were carried out in pure Hb A solution upon addition of potent allosteric effectors . This leads to low cooperativity and less than 100% oxygen saturation under room air at acidic pH . Under these conditions, mammalian or XL Hb have a maximum proton release at pH approximately 8 and an increased reverse Bohr effect . By contrast, a Root effect Hb exhibits a maximum proton release at neutral pH with the absence of reverse Bohr effect . Therefore the Root effect in fish Hb and the extreme stabilization of the T-state with effectors in mammalian Hb are not an identical phenomenon . Without crystallographic analyses of fish Hb exhibiting a Root effect, the molecular interpretation of this functional property still remains unexplained.

Grudn Khir, 1989 May-Jun, (3), 64 - 9
{Effects of low-frequency ultrasound on tumors cells in experimental animals}; Biriukov IuV et al.; The effect of low-frequency ultrasound was studied in different conditions in 197 in vitro experiments according to the histological form of the tumor . It was found that ultrasound with a frequency of 26.6--40.5 kHz, intensity of 2--8 Wt/cm2, and fluctuation amplitude of the end of the smooth cylindrical instrument-wave guide of 60--80 mcm caused destruction of the cells of malignant tumors of humans, whatever the histological form . Experiments with transplanted tumors--Zaidel's ascitic hematoma and sarcoma M-l of rats (202 rats) showed that after transplantation of a tumor cell suspension exposed to sonic waves for 5--7 minutes a tumor does not develop in highly susceptible experimental animals.

Med Phys, 1989 May-Jun, 16(3), 319 - 25
A two-fluid model for hematocrit distribution in microvascular networks; Hokkanen JE; A new theoretical network model for evaluating discharge hematocrits, explicitly based on plasma skimming at branches, is introduced . The particular network geometry chosen simulates bat wing microvasculature . Blood in vessels is approximated to be a two fluid with red cell suspension as the core and a plasma layer surrounding it . The plasma layer width depends on hematocrit, which leads to nonlinear hydrodynamic equations solved by iteration . Discharge hematocrit distributions are calculated by a computer for five generations of vessels . Dispersion of hematocrit values was found to be correlated to plasma skimming at branches . Contrary to previous suggestions, plasma skimming did not result in lowered mean hematocrit towards the capillaries . Network structure was found to be an important factor affecting the hematocrits . Mean discharge hematocrit remained steady against changes in vessel dimensions, capillary resistances, red cell concentration in plasma layer, and shape of the separation surface defining the streamlines entering the side branch . This high stable mean hematocrit is based on symmetry of the model network . Enhanced asymmetry tended to lower the hematocrit.

Brain Res Dev Brain Res, 1989 May 1, 47(1), 137 - 42
Pattern formation in the mammalian forebrain: patch neurons from the rat striatum selectively reassociate in vitro; Krushel LA et al.; Mechanisms involved in the developmental organization of the rat striatum were investigated in vitro . The neurons of the patch and matrix compartments were preferentially labeled in vivo with a {3H}thymidine injection on embryonic day (E) 13 or 18, respectively . Two or 7 days later the striatum was removed, dissociated into a single cell suspension and plated on a collagen-coated substrate . After 5 days in culture the neurons had migrated into aggregates . Within an individual aggregate, neurons labeled on E13 tended to clump together, whereas neurons labeled on E18 were randomly dispersed . Comparing between aggregates, {3H}thymidine-labeled E13 cells were located in aggregates containing numerous other labeled E13 cells, whereas {3H}thymidine-labeled E18 cells were dispersed randomly between aggregates . These results suggest that early born striatal neurons (primarily patch cells) selectively associate with each other, and that this process may be crucial to the developmental compartmentalization of the rat striatum.

Biull Eksp Biol Med, 1989 May, 107(5), 593 - 5
{Bone tissue formation in organ cultures of human bone marrow}; Luriia EA et al.; Bone formation in adult human bone marrow organ cultures is described . When culturing marrow fragments, thick bone lamina is formed . It has well-mineralized trabecular bone matrix with bone cells incorporated and is lined with osteoblast-like cells . In cultures of marrow deaggregated cell suspensions thin layers of the bone are only formed . Osteoclast-like cells develop in the cultures.

J Biomech Eng, 1989 May, 111(2), 152 - 6
Enhancement of heat transfer in red cell suspensions in vitro experiments; Carr RT et al.; New data on laminar heat convection with red cell suspensions have been gathered for both heating and cooling . When compared to data for the suspending medium alone, it is apparent that the red cells enhance laminar heat transfer when Pe greater than 4 . This is probably due to particle movements . These new data disagree with earlier studies which indicated no enhancement of heat transfer for blood cell suspensions . The data do agree with previous correlations for enhanced thermal transport in sheared suspensions.

In Vitro Cell Dev Biol, 1989 May, 25(5), 460 - 5
Epithelial cell interaction in air-liquid interface culture; Tchao R; A novel culture method has been developed to study the interaction of epithelial cells in the absence of a solid substratum . Starting with either a single cell suspension or aggregates, cells were floated at the interface of air and liquid culture medium . Two epithelial cell lines have been studied in this system: Madin-Darby canine kidney cells (MDCK), and a rat bladder tumor cell line (NBT-II) . Starting with a single cell suspension of MDCK, the floating cells coalesced in 24 h into sheets of cells . The cells were morphologically polarized with the apical surface facing the liquid medium . Domes were observed regularly in these sheets of cells . NBT-II cells migrated actively from aggregates at the air-liquid interface . In this floating culture, NBT-II cells produced extensive cell processes similar to those seen in cells grown on a solid surface . Because cells at the air-liquid interface lack a solid substratum for adhesion, cell membrane processes such as lamellapodia, retraction fibers, pseudopods, and long, intercellular connections can only exert a tension equal to or less than the surface tension of the liquid . Dimethyl sulfoxide 2% stimulated desmosome formation in floating NBT-II cells, resulting in a cribriform pattern in the sheet of cells . This method of interface can lead to new understanding of morphogenesis of epithelial cells, and the mechanism of cell motility and formation of cell processess.

Am J Physiol, 1989 May, 256(5 Pt 1), C1105 - 10
Extracellular Na+ electrode for monitoring net Na+ flux in cell suspensions; Brady HR et al.; A computer-linked extracellular sodium-sensitive electrode system is described that is suitable for the routine measurement of net Na+ transport in cell suspensions . The commercially available Na+ electrode exhibited high selectivity for Na+ over other cations and a rapid response time (less than 3 s) . This system resolved changes of 0.4 mM in the presence of 147 mM extracellular Na+ . Measurements of Na+ transport in suspensions of rabbit proximal tubules showed that ouabain caused a dose-dependent net Na+ influx with an inhibitor constant (Ki) of 2.5 +/- 0.2 microM and a maximal velocity (Vmax) of 229 +/- 7 nmol Na+.min-1.mg protein-1 . This compared favorably with the ouabain-induced K+ efflux (Ki = 2.4 microM; Vmax = 160 +/- 3.3 nmol K+.min-1.mg protein-1) and the ouabain-induced inhibition of respiration (Ki = 3.3 microM; Vmax = 11.8 nmol O2.min-1.mg protein-1) . In addition, Ba2+, a K+ channel blocker known to depolarize the cell, caused a net Na+ efflux, whereas glucose, a Na+-cotransported solute, promoted a net Na+ influx . This system should be a powerful tool for continuous monitoring of net Na+ fluxes in cell suspensions.

J Invest Dermatol, 1989 May, 92(5), 751 - 4
Relationship of sebaceous cell stage to growth in culture; Rosenfield RL; The maturational stage at which sebaceous epithelial cells are irreversibly committed to autolysis has not been determined in a biologic system . The purpose of these experiments was to determine the relationship of sebocyte maturation to their ability to grow in culture . Single cell suspensions from adult rat preputial glands were prepared, and sebocyte stages were classified using lipid staining as one criterion of maturation . Cells were subjected to one-step, isokinetic discontinuous density gradient centrifugation in Percoll . Cells were cultured on 3T3 feeder layers in an epithelial cell medium containing 20% fetal calf serum, growth factors, and antibiotics . Growth in culture occurred from sebocytes in all gradient fractions . The least growth was found from cells in the lightest density fraction (1.020), which contained sebocytes with the most lipid, i.e., the most mature . Over fivefold more growth occurred from cells in the most dense fraction (1.080), which contained undifferentiated and immature sebocytes . Cell growth in culture was then correlated with the type of sebocyte in each fraction plated . Although cell growth in culture correlated significantly with the number of undifferentiated cells (r2 = 0.460), epithelial colonies were found in the absence of discernible undifferentiated cells in most fractions . Growth in culture correlated much better with the number of early plus mid-differentiated sebocytes (r2 = 0.702) . These data suggest that, whereas mature sebocytes are not capable of attachment/proliferation, early differentiation of sebocytes is compatible with retention of the capacity to proliferate . If this property is shared by human sebocytes, it would contribute to the intransigence and diversity of acne-form lesions.

J Med Chem, 1989 May, 32(5), 1039 - 43
In vitro antiproliferative activity of 4-substituted 2-(2-hydroxyphenyl)thiazolines on murine leukemia cells; Elliott GT et al.; Two previously synthesized and two structurally novel thiazoline iron chelators are described . N4-Benzyl-N1,N8-bis{{2-(2-hydroxyphenyl)thiazolin-4-yl}carbonyl} homospermidine (5) proved to be the most potent antiproliferative and cytocidal compound in the series with in vitro IC50 values of 3 and 1 microM on L1210 and P388 murine cell lines . The N4-acetyl analogue 7 was considerably less active than 5 with IC50 and cell viability values that were similar to those of the structurally simple thiazolines 2 and 3 . The antiproliferative activity of 3 and 7 could be substantially reduced or ablated by delivery to cell suspensions as a 1:1 molar mixture with FeCl3, while the activity of 5 was unaffected by Fe(III) chelation . As expected, 3 induced a G1/S cell cycle block at the 100 microM block consistent with interference with DNA synthesis while 10 microM 5 did not affect L1210 cell cycle distribution . Tritiated thymidine incorporation studies confirmed that 5 was incapable of interfering with DNA synthesis at concentrations below 40 microM . Alkaline elution studies indicate that 5 does not cause DNA strand breaks in vitro at concentrations of 10 microM . The N4-benzyl group of 5 appears to impart in vitro potency as the N4-acetyl analogue 7 lacks comparable in vitro antiproliferative and cytocidal activity.

J Clin Invest, 1989 May, 83(5), 1651 - 60
Extracellular adenosine triphosphate activates calcium mobilization in human phagocytic leukocytes and neutrophil/monocyte progenitor cells; Cowen DS et al.; We have examined the ability of extracellular ATP to elicit intracellular Ca2+ mobilization in a broad range of human leukocytes at particular stages of hematopoietic differentiation . The average cytosolic {Ca2+} in various leukocyte populations was measured in Fura 2-loaded cell suspensions while the cytosolic {Ca2+} in individual, Indo 1-loaded leukocytes was assayed by flow cytometric methods . Utilizing normal blood- and marrow-derived cells, human leukemic cell lines, and mononuclear cell fractions derived from the blood of patients with various leukemias, we have found that ATP-induced Ca2+ mobilization appears restricted to leukocytes of neutrophil/monocyte ontogeny . Significant ATP-induced increases in cytosolic {Ca2+} were observed in neutrophils, monocytes, and myeloid progenitor cells as immature as myeloblasts, but not in lymphocytes . Extensive characterization of the ATP-induced changes in {Ca2+} observed in the HL-60 promyelocytic cell line have indicated these Ca2+-mobilizing effects of ATP can be correlated with an activation of inositol phospholipid breakdown via the occupation of P2-purinergic receptors Significantly, of the various agonists (FMLP, platelet-activating factor, LTB4, and ATP) which elicit equivalent and maximal Ca2+ mobilization in mature neutrophils and monocytes, ATP was the most efficacious stimulant of Ca2+ mobilization in immature neutrophil/monocyte precursors . Thus, expression of putative P2-purinergic receptors for ATP appears to precede expression of other receptor types known to activate the inositol phospholipid signaling cascades in terminally differentiated phagocytes.

Dev Biol, 1989 May, 133(1), 8 - 13
Determination of numbers of osteoprogenitors present in isolated fetal rat calvaria cells in vitro; Bellows CG et al.; When maintained in long-term cell culture in the presence of ascorbic acid and organic phosphate, single cell suspensions isolated from fetal rat calvaria form discrete, three-dimensional bone nodules . We have used limiting dilution analysis in microtiter wells to determine the number of osteoprogenitor cells expressing the capacity to form bone in the isolated mixed population, to examine the possibility of cooperativity among cell types in bone nodule formation, and to determine the effects of dexamethasone on osteoprogenitor cells . Cells plated at very low densities and screened for the presence or absence of bone nodules revealed a linear relationship (r = -00.997) between the number of cells plated and the number of bone nodules formed . The complete limiting dilution analyses showed that 1 of every 335 plated cells (0.30% of the cell population) has the capacity to form a bone nodule under standard culture conditions and when the actual numbers of nodules were quantitated from the same plated cell populations the ratio of nodules formed to plated cells was similar . Comparison of data from 13 different isolates of cells in which cells were plated into 35-mm dishes and number of nodules were determined indicated a mean +/- 95% confidence interval of one nodule for every 301 +/- 61 plated cells, consistent with the data obtained from the limiting dilution experiments . Dexamethasone increased the number of bone-forming cells to 1 in 225 cells, in contrast to 1 in 340 cells in the same population grown without added dexamethasone . The results suggest that approximately 0.30% of the cells in isolated rat calvaria populations are osteoprogenitor cells, that one osteoprogenitor cell gives rise to one bone nodule, that cooperativity between different cells in vitro is not necessary for bone formation, and that dexamethasone stimulates the expression of osteoprogenitor cells.

Exp Clin Endocrinol, 1989 May, 93(2-3), 187 - 92
Effect of serum from diabetes-prone BB/OK rats on neonatal rat pancreatic islets and islet cell suspensions; Schroder D et al.; Humoral-mediated cytolytic activity against neonatal rat pancreatic islet cells as well as islets of Langerhans was detected in sera of newly diagnosed diabetic BB/OK rats by measurement of enhanced 51Cr-release or insulin leakage . Beta-cell-specific functions of islet cell suspensions such as insulin secretion and (pro)insulin biosynthesis can be affected by BB rat serum in vitro.

Anticancer Res, 1989 May-Jun, 9(3), 799 - 803
Simultaneous detection of cellular ras p21 oncogene product and DNA content by two-parameter flow cytometry; Giordano M et al.; The rapid identification of the expression of oncogene products in specific cell types could be useful to investigate normal and malignant cell proliferation . We have developed a sensitive fixation - permeabilization technique (with 70% ethanol and 0.01% Triton x 100) for the detection of p21 ras oncoprotein and DNA content . Cell suspensions with negligible cell clumping, bright specific immunofluorescent staining were obtained with this method . Bivariate flow cytometry was then used to quantitate simultaneously the distribution of anti p21 ras oncoprotein (with a specific FITC-labeled antibody) and of total DNA (with propidium iodide) . The study was carried out in human leukemic cell lines HL-60 and K562, human breast carcinoma cell line MCF-7 and fresh neoplastic cells from human acute leukemia . The p21 ras oncoprotein was found in all phases of cell cycle . The degree of its expression, however, varied widely in diploid (C0/G1) cells from different samples, which could be related to differences in the relative proportion of G0 and G1 cells . Compared to the conventional gel electrophoretic technique, the use of bivariate FCM is feasible, fast, requires fewer cells per sample (2 x 10(6} and allows both the ras oncogene expression in intact cell populations as well as its relationship with cell cycle phases to be studied.

Res Commun Chem Pathol Pharmacol, 1989 May, 64(2), 299 - 316
Metabolic processes in isolated rat small intestine villus cells: effects of cis-diamminedichloroplatinum (II); Kralovanszky J et al.; Intestinal cells were isolated from male Fischer 344 rats by the collagenase portal vein perfusion procedure and evaluated for direct effects of cis-diamminedichloroplatinum (CDDP) and ethylacrylate (EtAc) on metabolic activities . Specific activities of marker enzymes of intestinal crypt and villus cells indicated that the preparations contained predominantly villus cells . Cell viability was generally greater than 90%, and was maintained longest when the cells were suspended in M-199 medium supplemented with 1% BSA . EtAc, an industrial intermediate which is toxic to tissues which are directly exposed to this chemical, had no apparent effect on rates of glucose metabolism or protein synthesis in suspensions of the isolated intestinal cells . These metabolic processes, however, were inhibited by the anticancer agent, CDDP; the mechanism of cytotoxicity of CDDP may therefore be due to interference with intermediary metabolism . The present studies indicate that isolated intestinal cell suspensions may be useful in examining direct and immediate effects of chemicals which are toxic to the intestinal epithelium, and in evaluating potential cytotoxic effects of CDDP analogs which have been developed.

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