Synthesis and biological activity of position 1 analogs of LH-RH.

(Formyl-sarcosine)1-LH-RH (I), (acetyl-sarcosine)1-LH-RH (II), (2-pyrrolidone-4-carboxylic acid)1-LH-RH (III), (N-methyl-2-pyrrolidone-4-carboxylic acid)1-LH-RH (IV, hydroxyproline1-LH-RH (V) and (cylcopentane-carboxylic acid)1-LH-RH (VI) were synthesized by solid phase methods on a benzhydrylamine resin support. Peptides I-IV were assayed for LH- and FSH-releasing activity over a 4-h period after subcutaneous infection into immature male rats in order to detect any prolongation ofactivity. The peptides were found to have the following integrated LH-releasing activities compared with LH-RH: I, 64%; II, 72%; III, 19%; IV, 58%. None of the peptides were found to be longer acting than LH-RH. Peptides V and VI were far less active, 0.001% and 1.4%, respectively.     

Presence and activity of alkaline phosphatase in two human osteosarcoma cell lines

The presence and activity of alkaline phosphatase in SAOS-2 and TE-85 human osteosarcoma cells grown in culture were examined at the ultrastructural level. A monoclonal antibody raised against purified human bone osteosarcoma alkaline phosphatase was used to localize the enzyme in cultures of the osteosarcoma cells. Similar cultures were analyzed for alkaline phosphatase activity using an enzyme cytochemical method with cerium as the capture agent. Alkaline phosphatase was immunolocalized at the light microscopic level in an osteogenic sarcoma and ultrastructurally on the SAOS-2 cell membrane and the enclosing membrane of extracellular vesicular structures close to the cells. In contrast, the TE-85 cells were characterized by the absence of all but a few traces of immunolabeling at the cell surface. Enzyme cytochemical studies revealed strong alkaline phosphatase activity on the outer surface of the SAOS-2 cell membrane. Much lower enzyme activity was observed in the TE-85 cells. The results support biochemical data from previous studies and confirm that SAOS-2 cells have a significantly greater concentration of alkaline phosphatase at the plasma membrane.     

Spontaneous pleural effusion in a human fetus.

Left sided pleural effusion was found in a human fetus spontaneously aborted at 20 weeks postmenstrual. Pulmonary hypoplasia associated with the effusion suggested that the fluid had been present for some time before delivery. Absence of other anomalies supports the view that the effusion was due to maldevelopment of the thoracic lymph channels.     

Immunocytochemical study of the GH cells in the anterior pituitary gland of human fetus II. Anencephalic fetus

In order to elucidate the effects of hypothalamic regulation on the morphology of GH cells, light and electron microscopic immunocytochemical examinations were carried out comparing GH cells in the anterior pituitary gland of anencephalic fetus with those of normal fetuses. Three types of GH cells were identified in the anterior pituitary gland of anencephalic fetus as well as in the normal fetus. Type-I is a small, round cell containing a few small secretory granules. Type-III is a large, polygonal cell with numerous large secretory granules. Type-II is a polygonal cell with medium-sized secretory granules. The Type-II GH cell was predominant in both anencephalic and normal fetuses. The most striking difference between anencephalic and normal fetuses was the presence of atypical forms of the Type II cell. These were polygonal cells containing secretory granules, which were either immunopositive or immunonegative to anti-human GH (anti-hGH) serum. Furthermore, two other types of GH cells were identified. The somatomammotroph (SM cell) contained GH and PRL in different granules within the same cell. Also, a different type of the GH cell was noted containing two varieties of secretory granules; one was immunolabeled only with anti-hGH and the other was not immunolabeled to either anti-hGH or anti-human PRL (anti-hPRL). From these results, we suggest that an absence of hypothalamic regulation in the anencehpalic does not seriously modify GH cell morphology but induces an altered GH storage pattern in some of the cells.     

Resistance to enzymic degradation of LH-RH analogues possessing increased biological activity.

Three analogues of LH-RH in which Dextrarotatory amino acids were substituted for the Gly⁶, and two additional analogues in which the Leu⁷ residue was also modified, were subjected to enzymic preparations derived from rat hypothalamus or anterior pituitary. These enzymes, known to cleave LH-RH, preferentially at the Gly⁶-Leu⁷ position, proved less effective in degrading all the analogues tested. Among the Gly⁶ substituted analogues, [D-Trp⁶] LH-RH, having the highest LH-releasing activity, was most resistant to degradation. Additional modification, at position 7, although rendering the analogues immune to enzymic attack, did not further enhance their biological potency. These data suggest that degradation of LH-RH is a physiological determinant of its biological activity and has therefore to be considered with on designing new, potent analogues of the hormone.     

Human B cell development. II. Subpopulations in the human fetus

In man, during fetal development the B cell populations show distinct phenotypes at different tissue sites. The pre-B and B lymphocytes of the fetal liver and bone marrow express IgM and B cell markers, B1 (CD20) and BA-1 (CD24). These "early" cells are negative with a number of other reagents, anti-IgD, RFB4 (CD22), RFB6 (CD21), and RFA-2, which on the other hand recognize peripheral B cells. These peripheral B lymphocytes in the developing fetus are heterogeneous. The diffusely distributed B cells in the earliest lymph node samples, 16 to 17 wk of gestational age, and from 16 to 21 wk in the spleen, are strongly IgM+ (IgD+,RFB4+,RFB6+, and RFA-2+) but lack T cell-associated markers such as T1 (CD5, p 67,000 dalton equivalent of murine Ly-1) and Tü-33. In fetal lymph nodes, primary nodules develop around the follicular dendritic (FD) cells from 17 wk onward, and contain a virtually pure population of B cells; B1+,BA1+,RFB4+,RFB6+,RFA-2+, which simultaneously express IgM,IgD together with T1 (CD5), a T cell-associated antigen. A sizeable subpopulation of these IgM+,T1+ cells are also positive for Tü-33, another T cell-associated marker. In the spleen, the B cells of the IgM+,IgD+,T1+ type appear in smaller numbers and only relatively late around wk 22. These cells are diffusely distributed at first, and start accumulating around the small FD cell clusters as soon as these emerge about the 23rd gestational wk. At that time, the IgM+,T1+B cells can also be washed out from the peritoneal and pleural cavities. The T1+,IgM+B cells may represent the normal equivalent cells of B chronic lymphoid leukemia and centrocytic lymphoma, and appear to be the counterpart of Ly-1+,IgM+B cells in the mouse.     

Synthesis and biological activity of LH-RH antagonists modified in position 1

As part of our studies on the design of more potent antagonists of the LH-RH (luteinizing hormone-releasing hormone) decapeptide, twelve new highly soluble D-Arg⁶-analogs have been synthesized. These peptides contain modifications in position 1 and are typified by the general formula (N-acetyl-X¹, D-p-Cl-Phe², D-Trp³, D-Arg⁶, D-Ala¹⁰) LH-RH. We have found that a lypophilic, aromatic substituent is required in position 1 in order to elicit antiovulatory activity at a dose as low as 3 μg. The larger the hydrophobic amino acid (X: p-Br-Phe, β-Nal-2) in position 1, the higher is the antiovulatory activity that can be attained. Analogs with non-aromatic or hydrophilic amino acids (X: Gly, Leu, Arg, His, Glu) in position 1 generally have much lower activities in this series of LH-RH antagonists.     

Applications of the extraction and of the radioimmunoassay of LH-RH in human urine]

The existence of endogenous LH RH like immunoreactivity is shown in human urines after appropriate extraction, bu the radioimmunoassay of LH RH. In normaly cycling and menopausal women the quantities of endogenous hormone found in urines are greater after acid extraction than those found after extraction at pH 7. Furthermore, the increase observed by extraction in acidified methanol is directly correlated and proportional to the quantity of hormone assayable by extraction at pH 7. The hypothesis of urinary excretion of LH RH as a polymer of immunoreactive units is suggested by this study.     

Hypersensitivity of the human gonadotrophs to the repeated administration of small doses of LH-RH.

The functional capacity of the gonadotrophs was assessed by repeated stimulation with small doses of LH-RH (5 microgram intravenously at 2-hour intervals for 3 injections) in normal women during the early and late follicular phases of the menstrual cycle. The results were compared to those obtained when a single dose (100 microgram) of the neurohormone was administered. During the early follicular phase, the release of LH and FSH remained about equal after the 3 successive injections of the small and after the large dose of LH-RH. During the late follicular phase, the release of LH and fsh increased progressively after the repeated administration of the 5 microgram of the neurohormone while the large dose induced a more pronounced and a more sustained pituitary response. This hypersensitivity of the gonadotrophs is observed when the E2 concentrations are higher than in the early follicular phase of the menstrual cycle.     

The role of N-acyl groups in the inhibitory activity of LH-RH analogues

Inhibitory analogues of luteinizing hormone-releasing hormone (LH-RH) were prepared with formyl-D-Trp¹, acetyl-D-Trp¹, valeryl-D-Trp¹, tartaryl-D-Trp¹, diacetyl-tartaryl-D-Trp¹, acetyl-Gly¹, and acetyl-Sar¹ successively replacing the position one in the analogue [D-Trp¹, D-p-Cl-Phe², D-Trp³, D-Phe⁶, D-Ala¹⁰]-LH-RH. The formyl-D-Trp¹ and acetyl-D-Trp¹ analogues yielded 100% blockade of ovulation at the 10 μg dose; the others were less potent and inhibited ovulation at the 50 μg dose. The inhibitory potency seems to correlate with the polarity of the acyl group.