Ultrastructural immunocytochemistry of glia cells

Summary The introduction of acrylate resins (Lowicryl K 4M, LR White) into electronmicroscopic immunocytochemistry applied to embedded tissue (post-embedding method) has improved the localization of antigens because of a satisfactory preservation of both ultrastructure and antigenicity of tissues. Here we describe a method that allows double staining of intracellular and membranous determinants in ultrathin sections of nervous tissue and cultures of peripheral nervous system cells. Ultrathin sections of the rat central nervous system fixed on uncoated grids were stained first for MBP selectively on the one face, then the opposite face was stained for GFAP using monoclonal antibodies and indirect immunogold staining method (IGS). Cultured Schwann cells induced to express major histocompatibility complex (MHC) class II antigens were stained for class. H antigens by pre-embedding method then followed by post-embedding IGS for the other intracytopasmic antigens.The Clinical Research Unit for Multiple Sclerosis is supported by Hermann and Lilly Schilling foundation     

Discriminant analysis of hematological and serum biochemical values in cynomolgus monkey (Macaca fascicularis) bred and reared under the indoor individually-caged conditions

The data on hematological and serum biochemical properties of laboratory-bred cynomolgus monkeys (Macaca fascicularis) at different ages were analyzed by discriminant analysis. All the animals had been bred and reared under uniform environmental conditions at Tsukuba Primate Center for Medical Science, N.I.H., Japan. The items used were as follows: red blood cell count (RBC), hematocrit value (Ht), hemoglobin concentration (Hb), mean corpuscular volume (MCV), white blood cell count (WBC), glutamic oxaloacetic transaminase activity (GOT), glutamic pyruvic transaminase activity (GPT), total protein concentration (TP), albumin concentration (ALB), albumin-globulin ratio (A/G), blood urea nitrogen (BUN), glucose concentration (GLU), total cholesterol concentration (TCHO), free cholesterol concentration (FCHO), triglyceride concentration (TG) and alkaline phosphatase activity (ALP). In total, 1086 animals in 10 age groups were examined. Data analyses were done with respect to the difference of sex. Discrimination was possible by Mahalanobis' generalized distance between centroids of groups. In canonical discriminant analysis (discriminant analysis with reduction of dimensionality), age was highly correlated to the value of the first canonical variate. From the approximate relative value of the eigenvector of the first canonical variate, the most discriminant variables are WBC, TP, ALB, A/G, TCHO, FCHO, TG, and ALP. It can be concluded that periodic measurement of these 8 parameters is necessary and sufficient to monitor the physiological conditions of growing monkeys.     

Effect of UV-B (290–320 nm) irradiation on growth and metabolism of cucumber cotyledons

Cucumber ( Cucumis sativus L. cv. Natsusairaku 3) seedlings were grown in a growth cabinet under UV-B (290–320 nm) irradiation (equivalent to the UV-B radiation normally incident at Tokyo, 36°N latitude, during clear sky conditions in mid-april on a weighted daily fluence basis) and a UV-B-free control condition. UV-B irradiation inhibited the growth of the cotyledons, i.e. the increase in area, and increase in fresh and dry weights of the cotyledons. The greatest inhibition rate was observed in the increase in area, causing a significant increase in specific leaf weight (the ratio of weight to area). UV-B irradiation had no significant effect on DNA and RNA contents in the cotyledons, but decreased protein content slightly. In contrast, the irradiation reduced the amounts of organic acids and soluble sugars, indicating that primary carbon metabolism was very sensitive to UV-B radiation. UV-B irradiation lowered the photosynthetic activity in the cotyledons without any effect on chlorophyll content and respiratory activity. These results indicate that UV-B radiation at the ambient level may act as a physiological stress in some UV-sensitive plants.     

Early and late periodic patterns of even skipped expression are controlled by distinct regulatory elements that respond to different spatial cues

We have identified the regulatory sequences required for the periodic expression of the Drosophila pair rule gene even skipped (eve). We find that the gradually changing pattern of periodic eve expression during early embryogenesis is directed by two distinct regulatory programs. Initially, eve expression in individual stripes is established by different regulatory elements, each of which responds to nonperiodic spatial cues provided, at least in part, by the gap genes. Later, coordinate expression of eve in all seven stripes is directed by a single regulatory region that responds to periodic cues provided by primary pair rule genes, including eve itself. As a consequence of this two-step regulatory program, eve functions both in the establishment of the periodic pattern of gene expression and in the subsequent specification of parasegmental boundaries.     

The structure ofHLA-B35 suggests that it is derived fromHLA-Bw58 by two genetic mechanisms

The primary structure ofHLA-B51 andHLA-Bw52 suggested thatHLA-B51 was derived fromHLA-Bw52 by the combination of a genetic exchange withHLA-B8 and a point mutation. To investigate the evolution of theHLA-B5 cross reactive group, theHLA-B35 gene was cloned and the primary structure was determined.HLA-B35 is identical toHLA-Bw58 except in the α₁ domain. The α₁ domain ofHLA-B35 except Bw4/Bw6-associated amino acids is identical to that ofHLA-B51 ^*, which was suspected to be an intermediate gene betweenHLA-B51 andHLA-Bw52. These data suggest thatHLA-B35 has evolved fromHLA-Bw58 in two steps; an in vivo replacement of the α₁ domain withHLA-B51 and genetic exchange with one of theHLA-Bw6 genes. These three genes andHLA-Bw58 are postulated to share a common ancestor.     

Existence of a polar digitalis-like factor in mammalian hypothalamus

We were able to partially purify a polar digitalis-like factor from rat and bovine hypothalami based on the capacity to inhibit [3H]ouabain binding to intact human erythrocytes. This factor was characterized in reference to the digitalis-like factor that we have isolated and reported on. Hypothalamic factor shared digitalis-like activities and physicochemical properties with the one derived from human urine and mammalian plasma. These findings strongly suggest that a polar digitalis-like factor identical to the circulatory factor does exist in mammalian hypothalamus.     

Endothelin-3 is a novel neuropeptide: isolation and sequence determination of endothelin-1 and endothelin-3 in porcine brain

The molecular forms of endothelin (ET) related peptides were investigated in porcine brain by using high performance liquid chromatography coupled with three specific radioimmunoassays. ET-1 and its oxidized form were isolated and sequenced as in the case of porcine spinal cord. A very small amount of big ET-1 (1-39) and its C-terminal fragment (big ET-1 (22-39] were also detected. Furthermore, immunoreactive (ir)-ET-3 was isolated and sequenced; its partial primary structure was identical to that of human (rat) ET-3. The concentrations of ir-ET-1 and ir-ET-3 in porcine brain were 140 fmol/g tissue and 5 fmol/g tissue, respectively. These results indicate that besides ET-1, ET-3 is a novel neuropeptide in the central nervous system.     

Presence of endothelin-1 in porcine spinal cord: isolation and sequence determination

We investigated the molecular forms of endothelin (ET) related peptides in porcine spinal cord by high performance liquid chromatography coupled with radioimmunoassays using three antisera raised against ET-1 and C-terminal fragments of ET-1 and big ET-1. ET-1 and its oxidized form were isolated as major immunoreactive peptides and sequenced. Furthermore, immunoreactivities like ET-3 and big ET-1(22-39) (contents: less than 8% and less than 1% of ET-1, respectively) were detected based on their chromatographic retention times and characteristics of immunoreactivity to the antisera. Big ET-1 was only scarcely detected. Immunohistochemical study showed the presence of ET-1-like immunoreactivity in motoneurons, dorsal horn neurons and dot- and fiber-like structures in the dorsal horn of lumbar spinal cord. These results indicate that ET-1 is present not only in endothelial cells but also in spinal cord, and that big ET-1 is converted into ET-1 in spinal cord by specific processing between Trp21-Val22. The data also indicate that ET-1 may act as a neuropeptide in the central nervous system.     

Conformational states of beta-lactamase: molten-globule states at acidic and alkaline pH with high salt

We present evidence that beta-lactamase is close to fully unfolded (i.e., random coil conformation) at low ionic strength at the extremes of pH and that the presence of salt causes a cooperative transition to a conformation with the properties of a molten globule, namely, a compact state with native-like secondary structure but disordered side chains (tertiary structure). The conformation of beta-lactamase I from Bacillus cereus was examined over the pH 1.5-12.5 region by circular dichroism (CD), tryptophan fluorescence, dynamic light scattering, and 1-anilino-8-naphthalenesulfonate (ANS) binding. Under conditions of low ionic strength (I = 0.05) beta-lactamase was unfolded below pH 2.5 and above pH 11.5, on the basis of the far-UV and near-UV CD and tryptophan fluorescence. However, at high ionic strength and low pH an intermediate conformation (state A) was observed, with a secondary structure content similar to that of the native protein but a largely disordered tertiary structure. The transition from the unfolded state (U) to state A induced by KCl was cooperative and had a midpoint at 0.12 M KCl (I = 0.17 M) at pH 1.6. A similar conformation (state B) was observed at high pH and high ionic strength. The transition from the alkaline U state to state B induced by KCl at pH 12.2 was cooperative and had a midpoint at 0.6 M KCl (I = 0.65 M). Light scattering measurements showed that state B was compact although somewhat expanded compared to the N state. The compactness of state A could not be determined due to its strong propensity to aggregate.(ABSTRACT TRUNCATED AT 250 WORDS)