Surface properties of irreversibly sickled cells differ from those of the bulk of sickle cells Studies by partitioning in aqueous phase systems

Counter-current distribution (CCD) of red blood cells (RBC) from individuaks with homozygous sickle cell (HbSS) disease in a charge-sensitive aqueous dextran-poly(ethylene glycol) phase system, which fractionates cells on the basis of surface properties, indicates that the percentage of irreversibly sickled cells (ISC) increases and the percentage of reticulocytes decreases with increasing cell partition ratios. The high partition ratios of ISC correspond to those of older RBC when RBC from normal individuals are subjected to CCD. Our results thus indicate that ISC differ in surface properties from those of the bulk of sickle RBC (including reticulocytes) in the population and that the difference is, most likely, charge-related. While the question as to whether ISC are indeed old cells has not yet been unequivocally answered, this view finds support in the fact that the independent parameters of ISC surface properties, as reflected by partition ratios, and densities correlate as they do in older RBC from normal individuals.     

Aerobic degradation of polychlorinated biphenyls by Alcaligenes sp. JB1: metabolites and enzymes

In contrast to the degradation of penta-and hexachlorobiphenyls in chemostat cultures, the metabolism of PCBs by Alcaligenes sp. JB1 was shown to be restricted to PCBs with up to four chlorine substituents in resting-cell assays. Among these, the PCB congeners containing ortho chlorine substituents on both phenyl rings were found to be least degraded. Monochloro-benzoates and dichlorobenzoates were detected as metabolites. Resting cell assays with chlorobenzoates showed that JB1 could metabolize all three monochlorobenzoates and dichlorobenzoates containing only meta and para chlorine substituents, but not dichlorobenzoates possessing an ortho chlorine substituent. In enzyme activity assays, meta cleaving 2,3-dihydroxybiphenyl 1,2-dioxygenase and catechol 2,3-dioxygenase activities were constitutive, whereas benzoate dioxygenase and ortho cleaving catechol 1,2-dioxygenase activities were induced by their substrates. No activity was found for pyrocatechase II, the enzyme that is specific for chlorocatechols. The data suggest that complete mineralization of PCBs with three or more chlorine substituents by Alcaligenes sp. JB1 is unlikely.Abbreviations PCB polychlorinated biphenyls - CBA chlorobenzoate - D di - Tr tri - Te tetra - Pe penta- - H hexa     

CD44 standard and variant isoform expression in normal human skin appendages and epidermis

 CD44 isoforms have been implicated in tumor progression and metastasis formation. This study presents a thorough immunohistochemical analysis of CD44 standard and isoform expression in normal human skin appendages and epidermis applying monoclonal antibodies against CD44s, CD44v3, -v4, -v5, -v6, and -v9. An improved immunohistochemical protocol with microwave-based antigen retrieval in paraffin sections and heavy metal amplification of the diaminobenzidine reaction product provided enhanced resolution and sensitivity as compared to studies on frozen sections. The hair follicle, the seborrheic and eccrine sweat glands were strongly positive for all CD44 isoforms studied. In the latter, the clear cells but not the dark (intercalated) cells were positive. The sudoriferous ducts adjacent to the glands were weakly positive for all CD44 isoforms and strongly positive near the skin surface. In the apocrine glands, the basal cells showed only a moderate positivity. The myoepithelial cells expressed only CD44s. In the epidermis, all CD44 isoforms were detectable, with strongest CD44 immunostaining in the lower third of the stratum spinosum and weaker staining in the stratum basale and the upper two-thirds of the stratum granulosum. The stratum granulosum and corneum were unreactive. Thus, a regional and cell type-specific CD44 expression was revealed. Accepted: 10 May 1996     

Retrieval of estradiol receptor in paraffin sections of resting porcine uteri by microwave treatment. Immunostaining patterns obtained with different primary antibodies

The unmasking of estradiol receptor in paraffin sections of Bouin's-fixed uterine tissue from ovariectomized gilts was attained with microwave treatment. Immunocytochemistry of the receptor was performed using a polyclonal or five monoclonal antibodies, two of which are commercially available, reacting with different domains of the protein and an amplified-peroxidase system for detection. With five of the antibodies, a predominance of nuclear staining was observed in cells of endometrial glands, while one monoclonal antibody (13H2), reacting with the receptor's domain E, showed a preference for the cytoplasmic receptor. In stroma, all antibodies detected more receptor in nuclei than in cytoplasm. In epithelium, the commercially available antibody H222, our monoclonals 13H2 and HT65, and the polyclonal antibody 402 demonstrated more receptor in cytoplasmic than in nuclear areas. In myometrium, the nuclei from longitudinal and ring muscles were definitely stained with the antibodies. We conclude that the accessibilities of the antibody epitopes of the receptor differ according to the functional uterine cell type.Dedicated to Professor Dr. Peter W. Jungblut on the occasion of his retirement     

Selection for β2-microglobulin mutation in mismatch repair-defective colorectal carcinomas

Novel peptide antigens complexed with human leukocyte antigen (HLA) and β₂-microglobulin (β2M) molecules are presented at the cell surface to cytotoxic T lymphocytes (CTLs), provoking lysis of the antigen-presenting cell [1]. In tumour cells, genetically altered or abnormally expressed proteins provide a source of peptides that can be presented to CTLs; the resulting anti-tumour CTL responses may provide part of the body's defence against cancer. Disabling mutations in the HLA and β2M proteins required for peptide presentation allow a tumour cell to escape destruction by CTLs. Cells with deficient DNA mismatch repair have high spontaneous mutation rates [2] and produce many altered proteins that are a potential source of numerous unique peptides. Mutator tumour cells might therefore be particularly vulnerable to immune surveillance and CTL attack. Mutator phenotypes [3] and [4] and loss of β2M (or HLA) expression [5] and [6] are both relatively common among sporadic colorectal tumours. We have compared the frequency of β2M mutations in sporadic colorectal and other tumours with and without a mutator phenotype. Mutations were more frequent among colorectal tumours with the microsatellite instability indicative of a defect in DNA mismatch repair. The inactivating β2M mutations were predominantly frameshifts, which is consistent with the underlying mismatch repair defects. Evasion of immune surveillance by acquiring β2M mutations therefore occurs at high frequency in tumour cells with a mutator phenotype due to defective DNA mismatch repair.     

Fitness consequences of helping behavior in the western bluebird

We examined the fitness consequences of helping behavior inthe western bluebird (Sialia mexicana) at Hastings Reservationin Carmel Valley, California, USA, and tested hypotheses forhow helpers benefit from engaging in alloparental behavior.Both juvenile and adult western bluebirds occasionally helpat the nest During a 12 year period, all adult helpers and mostjuvenile helpers were male. Helpers usually fed at nests ofboth their parents and rarely helped when only one parent waspresent. The frequency of pairs with adult helpers was only7%, but nearly one-third of adult males helped among those withboth parents on the study area. At least 28% were breeders whosenests failed. The propensity to help appears to depend uponparental survival, male philopatry, and the breeding successof potential helpers. Feeding rates were not increased at nestswith juvenile helpers, apparendy because breeding males reducedtheir feeding rates. In contrast, adult helpers increased theoverall rates of food delivery to the nest in spite of a reductionin the number of feeding trips made by both male and femaleparents. Helpers did not derive any obvious direct fitness benefitsfrom helping, but they had greater indirect fitness than nonhelpersdue to increases in nestling growth rates and fledging successat their parents' nests. Helpers fledged fewer offspring intheir first nests than did nonhelpers, suggesting that theywere birds with reduced reproductive potential. Although wehave not yet measured the effect of extrapair fertilizationson the fitness benefits of helping, we calculated the differencein fitness between helpers and nonhelpers as a function of thepotential helper's paternity when breeding independently andhis father's paternity in the nest at which he might help. Inconjunction with constraints on breeding and indirect fitnessbenefits, we predict that relatedness of males to the youngin their own as well as their parents' nests will influencehelping behavior in western bluebirds.     

Immunogold Localisation of the Intermediate Chain within the Protein Complex of Cytoplasmic Dynein

It was our goal to determine the location of the intermediate chain within the complex of cytoplasmic dynein by immunoelectron microscopy. To do so we generated two monoclonal antibodies (m74-1 and m74-2) specific for the intermediate chain. Both antibodies recognised the intermediate chain by sodium dodecyl sulphate–polyacrylamide gel electrophoresis immunoblot and ELISA assays of native and denatured proteins. When sucrose density gradient-purified cytoplasmic dynein from bovine brain was incubated with the gold-conjugated monoclonal antibodies, m74-1 and m74-2, and examined by negative staining, the gold label was found opposite the globular heads at the base of the V-shaped stalk of the motor complex. The labelling of the intermediate chain is the first mapping of a component within cytoplasmic dynein. The identification of the intermediate chain at the base of the complex supports a possible docking function of the intermediate chain.     

Product inhibition during the feeding phasein fed-batch ethanol fermentation of sugar-cane blackstrap molasses

Summary The following equations represent the influence of the ethanol concentration (E) on the specific growth rate of the yeast cells () and on the specific production rate of ethanol () during the reactor filling phase in fed-batch fermentation of sugar-cane blackstrap molasses: = ₀ - k · E and v = v ₀ · K/(K +E) Nomenclature E ethanol concentration in the aqueous phase of the fermenting medium (g.L^–1) - E_m value of E when = 0 or = 0 (g.L^–1) - F medium feeding rate (L.h^–1) - k empirical constant (L.g^–1.h^–1) - K empirical constant (g.L^–1) - M_as mass of TRS added to the, reactor (g) - M_cs mass of consumed TRS (g) - M_e mass of ethanol in the aqueous phase of the fermenting medium (g) - M_s mass of TRS in the aqueous phase of the fermenting medium (g) - M_x mass of yeast cells (dry matter) in the fermenting medium (g) - r correlation coefficient - S TRS concentration in the aqueous phase of the fermenting medium (g.L^–1) - S_m TRS concentration of the feeding medium (g.L^–1) - t time (h) - T temperature (° C) - TRS total reducing sugars calculated as glucose - V volume of the fermenting medium (L) - V₀ volume of the inoculum (L) - X yeast cells concentration (dry matter) in the fermenting medium (g.L^–1) - filling-up time (h) - specific growth rate of the yeast cells (h^–1) - ₀ value of when E=0 - specific production rate of ethanol (h^–1) - ₀ value of when E=0 - density of the yeast cells (g.L^–1) - dry matter content of the yeast cells