CYBEST Model 4. Automated cytologic screening system for uterine cancer utilizing image analysis processing

CYBEST (Cyto-Biologic Electronic Screening System) utilizes image analysis technology for the automated prescreening of cervical cytology specimens. CYBEST Model 3, which includes a television scan system and automatic shading control, achieved our initial goal of rapid specimen processing (no more than three minutes to achieve a final specimen assessment). This paper describes CYBEST Model 4, developed in 1981; with the minicomputer of Model 3 replaced by a microcomputer, Model 4 is considerably smaller, about the size of a business desk. A new parameter, the intranuclear configuration (chromatin pattern), was added to the four parameters used in Model 3. The five parameters now used for the assessment and ranking of cytologic abnormalities are nuclear size, nuclear-cytoplasmic ratio, nuclear optical absorption, nuclear shape and intranuclear configuration. The other features of Model 4 are almost the same as those of Model 3. As an optional function, individual parameter measurement data, assessment of atypicality grade and cell images can be displayed on the CRT monitor by pointing to a cell with a light pen system. After completion of screening of a specimen, the ten cells judged to be most abnormal can be called automatically into the microscope optical field or the CRT monitor (in order ranging from the cell with the highest atypicality rank down) along with their associated data and the system's assessment of the specimen. By connection to a small business computer, all data can be transferred to a floppy disk for later retrieval.     

CRF-containing neurons of the rat hypothalamus

Summary The immunoreactive CRF-neurons of the rat hypothalamus have been examined immunohistochemically employing anti-rat CRF serum. These neurons are confined to the paraventricular nucleus, dorsomedial-lateral hypothalamic area, and suprachiasmatic nucleus, and are, respectively, also immunoreactive to anti-Met-enk, -alpha-MSH, and -VIP sera. Intraventricular administration of colchicine (50 g/5 l/rat) induces a dramatic enhancement of the immunostainability of the cell somata, and also accelerates the development of immunoreactivity of other stored peptides, especially in the paraventricular nucleus.The CRF-neurons respond to adrenalectomy by showing increased immunoreactivity and an increase in the number of cell bodies; in the dorsomedial-lateral area and suprachiasmatic nucleus, there is also an enhanced immunoreactivity for alpha-MSH and VIP, respectively. CRF-cells in the paraventricular nucleus become markedly hypertrophied, but do not show any enhanced immunoreactivity for Met-enk. Since the axons of the paraventricular neurons run to the median eminence, it is probable that they are involved with the endocrine control of hypophysial ACTH release. It is concluded that the CRF-containing neurons in rat hypothalamus consist of three types which are functionally and morphologically different.     

Histochemical and cytochemical demonstration of Ca++-ATPase activity in the stellate cells of the adenohypophysis of the guinea pig

Summary The histo- and cytochemical localization of Ca⁺⁺-ATPase activity in the adenohypophysis of the guinea pig was studied utilizing a newly developed method (Ando et al. 1981). An intense reaction was observed in the wall of the blood vessels and between non-secretory cells (stellate cells) and endocrine cells of the pars distalis. Under the electron microscope the Ca⁺⁺-ATPase reaction product was located extracellularly in relation to the plasmalemma of the stellate cells. This reaction was dependent on Ca⁺⁺ and the substrate, ATP, and reduced by the addition of 0,1 mM quercetin to the standard incubation medium. Preheating of the sections before incubation completely inhibited the enzyme activity. When Mg⁺⁺ in different concentrations were substituted for Ca⁺⁺ in the incubation medium the reaction was always reduced. Both Ca⁺⁺ and Mg⁺⁺ in the incubation medium also reduced the reaction. The plasmalemma of the endocrine cells contains no demonstrable amount of Ca⁺⁺-ATPase activity. The function of the Ca⁺⁺-ATPase activity is discussed in relation to the regulation of the extracellular Ca⁺⁺ concentration which seems to be important with respect not only to the secretory process of the endocrine cells but also to the metabolism of the adenohypophysis.     

Purification of viral proteins from avian sarcoma virus QV2.

A procedure was established whereby most of the major viral proteins were isolated to apparent homogeneity in biologically and immunologically active forms from a single batch of avian sarcoma virus QV2. For the initial step of purification, gently disrupted virions were fractionated by CsCl centrifugation into envelope proteins, RNA-dependent DNA polymerase, and viral core proteins. Further purification of envelope glycoproteins and DNA polymerase was performed by affinity chromatography on agarose columns cross-linked with plant lectins and poly(C), respectively. On the other hand, core proteins were fractionated by a combination of gel filtration and ion-exchange column chromatography into components p27, p19, and p15. The core protein p15 thus isolated retained proteolytic activity even after storage for 6 months. The present study also demonstrated that QV2 p19 is structurally altered from the corresponding protein of avian myeloblastosis virus (AMV), a reference avian leukosis-sarcoma virus having a well-characterized polypeptide composition.     

The cytoskeleton-dependent localization of cdc2/cyclin B in blastomere cortex during Xenopus embryonic cell cycle

In the early development of the frog, Xenopus laevis, blastomeres undergo synchronous divisions at about the 12th cell cycle, followed by asynchronous divisions, which is referred to as mid-blastula transition (MBT). We investigated the distribution of several regulating factors for cell cycles around MBT using immunocytochemistry and confocal fluorescence microscopy. At the 8th cell cycle, most of the cdc2/cyclin B was localized in the cortical cytoplasm throughout the cell cycle, in the centrosomes and the nucleus at interphase and prometaphase, and in the spindles at metaphase and anaphase. Cdc2 was also localized in the chromatins at metaphase and anaphase. Cyclin B1 mRNA was localized in the periphery of the nucleus, but not in the cell cortex. At the 13th cell cycle, the amount of cdc2/cyclin B in the cortical cytoplasm decreased, and the inactive form of cdc2, phosphorylated at tyrosine 15, appeared in the nucleus and the centrosomes at interphase, indicating that the regulation of cdc2 by phosphorylation occurs around MBT. When the blastomeres were treated with nocodazole or latrunculin A at the 8th cell cycle, the amount of cortical cdc2 decreased, but that of cyclin B did not change. The cortical localization of cdc2 is dependent upon both microtubules and microfilaments. Most of the cdc27 was localized in the centrosomes, and in the spindle poles, but no significant difference was observed between the 8th and the 13th cell cycles. It is possible that the cortical MPF activity is regulated by the differential localization between cdc2 and cyclin B.     

Oxysterols increase in diabetic rats

To address whether diabetes enhances lipid peroxidation and attenuates nitric oxide (NO) generation resulting in tissue complications, we measured oxysterols and NO metabolites (NOx) in the tissues of diabetic Wistar rats. After 4 weeks of streptozotocin injection (STZ, 80 mg/kg, i.p.), we measured 7 alpha- and 7 beta-hydroperoxycholest-5-en-3 beta-ol (7 alpha-OOH and 7 beta-OOH), 7 alpha- and 7 beta-hydroxycholesterol (7 alpha-OH and 7 beta-OH) and 7-ketocholesterol (7-keto) by HPLC in the kidneys, heart, and liver. All the oxysterols were much higher in the diabetic than in sham rats, while the extent of the increase was higher in the order of the kidney, heart, and liver. Together with high blood urea nitrogen, the data indicate that the kidney is the predominant target of early diabetic complications. Plasma NOx were decreased by 20% in the STZ rats. The enhanced oxidative stress in diabetes would increase oxysterols by peroxidation, while superoxide is known to reduce NO by reaction to form another potent oxidant peroxynitrite.     

Accelerated impairment of spermatogenic cells in SOD1-knockout mice under heat stress

For normal spermatogenesis, the temperature of the scrotum is lower than that of the body. The mechanism by which mammalian testes undergoes cell death as the result of exposure to heat continues to be a matter of debate. Since generation of reactive oxygen species (ROS) during heat stress and involvement in spermatogenic cell damage are postulated, we induced experimental cryptorchidism in the testes of SOD1-knockout mice and examined effects of the gene deficiency. The cleavage of DNA in testicular cells, as judged by TUNEL staining, were elevated in SOD1-knockout mice at an earlier stage than in the wild-type mice. To confirm responsiveness of SOD1 for this high susceptibility to heat stress, spermatogenic cells were isolated from SOD1-knockout and wild-type mice and cultured at 32.5 and 37°C. The cells isolated from SOD1-knockout were more vulnerable at both temperatures than those from wild-type mice. The exposure of cultured rat spermatogenic cells to ROS induced the release of cytochrome c from mitochondria, while Sertoli cells were more resistant under the same conditions. Tiron, a superoxide scavenger, suppressed the heat-induced release of cytochrome c from mitochondria. Collectively, these data suggest that ROS are generated during heat stress and cause spermatogenic cell death. Alternatively, since even a short exposure triggers harmful damage to spermatogenic cells, generated ROS may function as a type of signal for cell death rather than directly causing oxidative damage to cells.     

Glucan-binding activity of silkworm 30-kDa apolipoprotein and its involvement in defense against fungal infection

The silkworm Bombyx mori 30-kDa lipoproteins (6G1 and 19G1), major components of the hemolymph, were shown to bind to glucans. 6G1 apolipoprotein was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and assayed for its binding activity. The purified recombinant 6G1 apolipoprotein specifically bound to beta-glucan, but not to chitin, mannan, peptidoglycan, or oligosaccharide chains on glycoproteins. The beta-glucan binding of the recombinant 6G1 was inhibited by laminaribiose and laminarin, a soluble glucan, but not by lipopolysaccharide or insect blood sugar, trehalose at physiological concentration. Furthermore, the recombinant 6G1 was shown to participate in the activation of prophenoloxidase cascade and to interfere with hyphal growth of the entomopathogenic fungus Paecilomyces tenuipes, injected into pupae of B. mori. These results suggest that 6G1 lipoprotein plays a role in the protection of B. mori against invading fungi.