Karl W. Lanks

The relationship between human papillomavirus and lower genital intraepithelial neoplasia in immunosuppressed women

In a group of 20 immunosuppressed women with lower genital neoplasia, evidence of associated human papillomaviral infection was found in all patients on the basis of the histologic identification of koilocytes in the upper strata of areas of mild or moderate dysplasia. Immunohistochemical study of similar areas disclosed human papilloma structural antigens in the lesions in 60%, while 50% had lesions in which human papilloma virions were detected by the electron microscope. An abnormal immunologic status, indicated by an altered T-helper/T-suppressor ratio, a deficient response to mitogenic stimulation, or both, was confirmed in 80% of the patients studied. Twelve of the 20 patients had unusually persistent and recurrent intraepithelial neoplasia, and in one the disorder progressed to invasive epidermoid carcinoma. The progressive behavior of human papillomavirus-associated neoplasia in these immunosuppressed patients might represent an accelerated version of the long-term course of such lesions in immunocompetent hosts.

Cevalent attachment of lactoperoxidase to polystyrene tissue culture flasks

Abstract In the present study a method is developed whereby lactoperoxidase is covalently coupled to polystyrene tissue culture flasks and is used to radioiodinate monolayer cell proteins that come into intimate contact with the surface. Coupling was effected by the reaction sequence: (i) nitration using HNO 3 H 2 SO 4 , (ii) reduction with sodium hydrosulfite, (iii) simultaneous incubation with lactoperoxidase and dimethylsuberimidate dihydrochloride. Each step was monitored by differential infrared spectroscopy. Either enzyme activity or total protein binding could be optimized. Iodination of intact mouse L cells attached to this surface was efficient, and the distribution of labeled trichloroacetic acid-precipitable material indicated preferential incorporation into extracellular sites.

Effects of low molecular weight nutrients on the pattern of proteins synthesized by non-proliferating murine L cells.

Abstract When L cells are cultured for prolonged periods of time without a medium change, the rate of synthesis of two polypeptides (82 000 and 95 000 mol. wt) is greatly increased while that of two other polypeptides (85 000 and 56 000 mol. wt) is correspondingly decreased. These changes in protein synthesis pattern were previously shown [1] to be due to depletion of low molecular weight nutrients from the medium. The present study shows that although the medium is severely depleted of glucose and several amino acids, glucose depletion alone is solely responsible for induction of the observed phenomena. Increased synthesis of the 95 000 and 82 000 mol. wt polypeptides has been shown by other investigators to occur in a different cell system under conditions of glucose deprivation. The present study shows that the synthesis of two additional major cytoplasmic proteins, the 85 000 and 56 000 mol. wt polypeptides, is also glucose regulated.

Spontaneous reactivation of acetylcholinesterase following organophosphate inhibition: I. An analysis of anomalous reactivation kinetics

The first kinetic studies on the spontaneous reactivation of Sarin-inhibited acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) are reported. With increasing pH the extent of reactivation increases while the observed rate constant decreases. An analysis of the change in aging rate constant as a function of pH suggests that the aging of alkyl-alkoxy phosphonylated acetylcholinesterases is not solely acid catalyzed.

INHIBITION OF GLUCOSE-REGULATED AND HEAT SHOCK PROTEIN INDUCTION BY LOW TEMPERATURE

The present study evaluating induction of the major stress proteins in the subphysiological temperature range (25-33 degrees C) shows that none of the agents used could effectively induce the heat shock proteins (hsp) or the glucose related protein grp95 at low temperature. However, grp82 was still induced by some amino acid analogs and by glucose deprivation while certain oxygen-regulated proteins were still induced by hypoxia at 25 degrees C. Analogs were incorporated and protein turnover was increased at low temperature even though most stress proteins were not induced. Synthesis of hsps, but not that of grps, was induced if cultures containing analog-substituted proteins were shifted to 37 degrees C. Temperature dependence of hsp induction by arsenite showed a sharp threshold between 30 degrees C and 33 degrees C. Low temperature inhibition of induction points to the existence of a temperature-dependent mechanism operating within the normal physiological temperature range and may be a useful parameter in evaluating proposed mechanisms of stress protein regulation.

Patterns of proteins synthesized by non-proliferating murine L cells.

Abstract When L cells were plated at high density (2 × 105/cm2), proliferation ceased within 3 days, but the cells remained viable and capable of reinitiating DNA synthesis for at least 16 days. SDS-polyacrylamide gel electrophoresis of [35S]methionine labeled polypeptides at various intervals after plating revealed distinct changes in the relative abundance of major cellular proteins beginning 9 days after DNA synthesis ceased. An 84 000 mol. wt polypeptide increased markedly in amount while a polypeptide of 94 000 mol. wt disappeared. Autoradiograms following pulse-labeling showed that these changes were due to increased synthesis of the 84 000 mol. wt polypeptide and decreased synthesis of the 94 000 mol. wt polypeptide. Increased synthesis of a 109 000 mol. wt polypeptide occurred without a concomitant change in its relative abundance. These alterations in the pattern of proteins synthesized revert to normal after feeding with serum-free medium even though DNA synthesis does not resume. Therefore, it appears that even though no abrupt changes in the synthesis of major cellular proteins occurred upon cessation of proliferation, eventually a distinct adaptive pattern of protein synthesis develops in response to the changing culture conditions.

Purification and characterization of a major component from the cytoplasmic matrix of cultured murine L cells

This study describes the purification and preliminary characterization of a 94 000 molecular weight protein (S-94) previously known only as a major band in SDS-polyacrylamide gels of total proteins from cultured murine cells. Native S-94 is a soluble constituent of the cytoplasm and sediments at 5.0 S in sucrose gradients, behavior compatible with that of a 94 000 molecular weight monomer. S-94 is purified more than 20-fold from the 100 000 X g supernatant of murine L or L1210 lymphoma cells by DEAE-Sephadex (A-50) chromatography in the presence of 2-mercaptoethanol followed by Sephadex G-200 chromatography in 8 M urea. The protein prepared by this procedure is extensively aggregated, but is essentially homogeneous by SDS-polyacrylamide gel electrophoresis. S-94 preparations from the two types of murine cells behave identically during the purification procedure and are presumed to be the same protein. It appears that these cells contain only one major protein of 94 000 molecular weight. Purified S-94 yields a distinctive pattern of fragments following CNBr degradation, including one peptide of 36 100 molecular weight. The amino acid composition is distinguished by being relatively rich in threonine, glycine and lysine, but poor in valine, leucine and tyrosine.

Elimination ofM. hyorhinis from murine neuroblastoma cell lines by in vivo passage

SummaryCell lines derived from a murine neuroblastoma, Clone N18, were cured of theirM. hyorhinis infection by in vivo passage. The major variable determining success of this method was found to be the incubation time in vivo. Infected cells maintained in vivo for 27 d or more and then placed in culture were free of mycoplasma whereas those maintained in vivo for 7 or 14 d were found to still be infected. This approach to eliminating mycoplasma infection may be successful using other tumor cell lines.

Spontaneous reactivation of acetylcholinesterase following organophosphate inhibition. II. Characterization of the reactivating components.

Repeated cycles of inhibition by a variety of organophosphates followed by spontaneous reactivation reveal a component of electric eel acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) which preferentially reactivates. That the observed enzymatic activity truly resides in acetylcholinesterase is indicated by its sensitivity to a specific inhibitor and by molecular weights for subunits and native enzyme which are approximately the same as those for the major fraction of enzymatic activity which behaves in the classical manner. The Km values for phenyl acetate of the two components are similar but the rate constant for covalent bond formation, k2, with isopropyl m-nitrophenyl methylphosphonate is greatly reduced in the spontaneously reactivating species. The molecular basis for these observations is discussed.

Lactoperoxidase-catalyzed iodinaton of cell cultures infected with mycoplasma

SummaryHamster BHK cells or secondary cultures of mouse embryo fibroblasts are not iodinated by lactoperoxidase in the absence of hydrogen peroxide. When such cell cultures are infected with a noncultivable strain ofM. hyorhinis, endogenous peroxide generation is sufficient to permit nearly maximal iodination. The SDS-polyacrylamide gel pattern of iodinated cell surface polypeptides is essentially the same regardless of the source of peroxide and whether or not the cultures are infected with mycoplasma.