Topographical and ontogenetic study of the neurons producing growth hormone-releasing factor in human hypothalamus

Neurons producing growth hormone-releasing factor have been characterized and analyzed by immunohistochemistry in the hypothalami of human fetuses, neonates, infants and adults, using two antibodies against human pancreatic GRF (hpGRF). One of the antibodies recognized both the hpGRF(1-40)OH and hpGRF(1-44)NH2 in the mid portion (between the 28th and 39th amino acid), the other one specifically recognized the C-terminal end of hpGRF(1-44)NH2. These two antibodies stain a single neuronal system with cell bodies mainly located in the infundibular (arcuate) nucleus, and in the ventromedial and lateralis tuber nuclei. These neurons project to the median eminence where they give numerous endings in contact with portal vessels. These neurons are distinct from those containing LH-RH, somatostatin, CRF or pro-opiocortin. In fetuses, neurons immunoreactive with hpGRF antibodies are first detected at the 29th week. They display a neuroblastic aspect which persists after birth. Immunoreactive fibers are detectable in the median eminence after the 31st week. These results demonstrate that the infundibular nucleus plays a major role in control of GH secretion in man and that secretion of GRF appears late during fetal life; this suggests that the first stages of differentiation and development of GH producing cells in the human fetus do not depend on hypothalamic GRF secretion.     

Ontogeny of the response to growth hormone-releasing factor

Primary cell cultures were prepared from fetal, neonatal and adult rat pituitaries and evaluated for their ability to secrete growth hormone (GH) in response to growth hormone-releasing factor (GRF). Pituitary cells prepared from fetuses at days 19 and 21 of gestation, neonatal animals at the day of birth (day 0) or the following day (day 1) and peripubertal male rats showed full dose response curves to GRF with maximal GH release when stimulated with 1 X 10(-10) M rat GRF. At this concentration of GRF, the amount of GH released was not different from that elicited by activation of adenylate cyclase with 1 X 10(-5) M forskolin. In contradistinction, a preparation of cells from fetuses at day 18 of gestation did not show the same release of GH when challenged with 1 X 10(-10) M GRF and forskolin (0.057 +/- 0.001, compared to 0.076 +/- 0.003 micrograms/10(5) cells per 4.5 h), although the cells clearly responded to both secretagogues (basal levels of GH, 0.029 +/- 0.002 micrograms/10(5) cells per 4.5 h). While cells prepared from fetuses at day 21 of gestation or from animals after birth released 5-10% of their total cellular GH content, those prepared from 18- and 19-day fetuses released as much as 40% of their total GH suggesting there is a maturation of intracellular GH processing that occurs late in gestation. The results show that, in late pregnancy, the rat fetal pituitary is highly responsive to growth hormone-releasing factor and suggest that this peptide participates in regulating GH levels during the perinatal period.     

Photoreceptor membrane breakdown in the spider Dinopis: GERL differentiation in the receptors

Summary In the spider Dinopis, retinae of the posterior median eyes synthesise new photoreceptor membrane in bulk at dusk and destroy it at dawn (Blest, 1978). During the dawn period, there is a rapid, anticipatory differentiation of unusual organelles from the rough endoplasmic reticulum (RER) in the intermediate segments of the receptors. This system is classified as GERL. Its products appear to play a role in the autolysis of photoreceptor membrane. Consistent topographical relationship to Golgi bodies has not been determined. Circumscribed regions of RER whorls first reorganise to yield fenestrated profiles; these differentiate further to a number of structures by condensation and loss of ribosomes. Smooth tubular profiles are termed rigid tubules to indicate their probable homology with the rigid lamellae of vertebrate secretory cells. More complex smooth multilocular bodies are also produced. Evidence is discussed which implies that both rigid tubules and multilocular bodies give rise to condensing vacuoles. These, in turn, pinch off coated vesicles assembled as Nebenkerne. Some rigid tubules are transported to the interrhabdomeral cytoplasm of the receptive segments. At late stages of differentiation, rigid tubules, multilocular bodies and Nebenkerne give strong, positive responses to zinc iodide-osmium tetroxide (ZIO) treatment; early stages and both cis and trans Golgi components do not. GERL differentiation is independent of immediate illumination of the retina at dawn. It is suggested to mediate the lysis of membrane degradation products by the production of hydrolases.We thank Professor D.T. Anderson F.R.S. for our use of field facilities at the Crommelin Biological Field Station of Sydney University at Warrah, Pearl Beach, N.S.W., and Andrew and Sally Austin and Sally Stowe for help in the field. We are indebted to Rod Whitty and the Electron Microscopy Unit for advice and support throughout these studies     

Modulation of drug permeability in Chinese hamster ovary cells. Possible role for phosphorylation of surface glycoproteins.

We have previously reported that the uptake of colchicine and other drugs in Chinese hamster ovary (CHO) cells can be greatly enhanced by the addition of metabolic inhibitors such as cyanide (See, Y.P., Carlsen, S.A., Till, J.E. and Ling, V. (1974) Biochim. Biophys. Acta 373, 242-252). This has led us to postulate the presence of an active drug permeability barrier in these cells. In this paper we provide evidence for the dependence of this permeability barrier on intracellular ATP levels. Colchicine-resistant mutants of CHO cells exhibiting a reduced drug permeability, however, can maintain this drug permeability barrier at much lower ATP levels, suggesting that they possess an altered active drug permeability barrier. We have also observed a membrane-associated protein kinase-phosphoprotein phosphatase system in the isolated membranes of mutant and wild-type cells. Differences in the intrinsic protein phosphorylation patterns between the membranes of these cells have led us to conclude that the control of the drug permeability barrier may be mediated via the phosphorylation of at least two high molecular weight surface glycoproteins.     

Endorphins are located in the intermediate and anterior lobes of the pituitary gland, not in the neurohypophysis.

Immunocytofluorescence techniques with well characterized anti-sera to α-endorphin and β-endorphin show presence of these two peptides in all cellular elements of the pars intermedia of the rat hypophysis, and in discrete cells of the pars distalis (adenohypophysis) at the complete exclusion of the neurohypophysis (pars nervosa, posterior lobe).     

Differentiation of aflatoxins from territrems.

Three methods were adopted for differentiation of aflatoxins B1 and B2 from territrems A and B. They were as follows. (i) Then-layer chromatography coupled with chemical confirmation. A significant decrease in the Rf value of trifluoroacetic acid-treated aflatoxin B1 developed in chloroform-acetone (85:15, vol/vol) was satisfactory in differentiating this toxin from the other three. (ii) High-pressure liquid chromatography monitored synchronously at two wavelengths, 365 and 335 nm. The ratio derived from this double-wavelengh detection could serve as an indicator of the presence of each toxin. (iii) Velasco's flurotoxin meter method, which is used for the determination of aflatoxins within the range of 0 to 50 ng/ml, was not significantly affected by territrems even when they were present in quantities at the microgram-per-milliliter level.     

精河株斑点热立克次体在非洲钝缘蜱体内的分布

本文报告用乙二醇甲基丙烯酸(GMA)包埋、切片和姬姆萨染色观察精河株斑点热立克次体在非洲钝缘蜱(Ornithodoros moubata)体内的分布情况。接种立克次体后2天,即可从蜱的血淋巴涂片中查见立克次体,但各器官和组织的切片6天后才开始出现阳性,尤以中肠最迟。叮咬感染的蜱的中肠感染较早也较严重。无论接种或叮咬感染,除睾丸和精子外,所查的器官和组织包括涎腺、基节器、中肠、直肠、卵巢以及雄蜱的附腺和贮精囊等切片中都查见立克次体。有的蜱感染很严重,立克次体充满了许多器官和组织的细胞。     

非洲钝缘蜱传播精河株斑点热立克次体的途径

非洲钝缘蜱(Ornithodoros moubata)叮咬感染精河株斑点热立克次体的豚鼠后45-52天,再叮咬正常豚鼠,可使后者感染。除个别蜱外,受精的感染雌蜱第一次产的卵(吸感染血后10—19天产出)和1龄稚蜱均为阴性。间隔.1个月又喂以正常豚鼠血,这些蜱第二次的吸血率、产卵数和卵的孵化率都明显降低,但从第二次六的卵和1龄稚蚌压印片中均查见大量立克次体。经卵传递率60%,子代感染率59%(6-100%)。     

Somatic embryogenesis and plant regeneration in an interspecific hybrid of Oryza

An embryogenic callus was obtained from immature panicle of an interspecific hybrid (Oryza sativa x O. latifolia) F₁. The medium consisted of HE salts supplemented with 2,4-D, NAA (each 2 mg/l), kinetin (3 mg/l), yeast extract (1360 mg/l) and casein hydrolyzate (300 mg/l). The callus was milk-white in colour compact and granulate in texture. Various developmental stage of embryoid, such as globular, heart-shape, scutellum-shape and mature embryoid were observed in an embryogenic callus. Plantlets were successfully regenerated from 1-month-old callus with more than 80% regenerational frequency in each subculture for 12 passages.     

Isolation and characterization of a succinimide variant of methionyl human growth hormone.

Deamidation of asparagine and glutamine residues, isomerization of aspartic acid side chains, and racemization of the L- to the D-form of the amino acids are common spontaneous chemical reactions known to occur in proteins. Previous studies have implicated succinimides as intermediates in these reactions; however, the evidence has been indirect. Our results demonstrate, for the first time, the presence of a succinimide intermediate in an intact protein. The succinimide (cyclic imide) variant was isolated from thermally stressed recombinant methionyl human growth hormone (hGH) by high performance anion-exchange chromatography, further purified by reversed-phase high performance liquid chromatography, and analyzed by tryptic mapping. A later eluting tryptic peptide, compared with the native T12 peptide (residues 128-134, Leu-Glu-Asp-Gly-Ser-Pro-Arg), was analyzed by mass spectrometry (MS). This variant had a protonated molecular mass of 755.3 atomic mass units (u), as compared with 773.3 u for the native T12 peptide. A difference of 18 u, a loss of water, is consistent with the formation of a succinimide intermediate at Asp-130 of methionyl hGH. MS/MS analysis of the cyclic imide-containing peptide verified that the modification occurred at Asp-130. A difference of 18 u was also observed for the intact cyclic imide methionyl hGH variant (22,238 u), as measured by electrospray mass spectrometry, compared with native methionyl hGH (22,256 u).