From paper chromatography to drug discovery: Zaffaroni.

A paper chromatography assay for adrenocortical steroids, published just as cortisone's therapeutic value in arthritis was announced, had far-reaching consequences. Its use by Upjohn scientists led to the first synthesis of cortisone. Interest in the assay also led the author to an association with Syntex S.A. in Mexico, which he was instrumental in transforming from a small chemical company into the large pharmaceutical firm, Syntex Corporation, having its base of operations in the United States. The major events of this history are described, showing how steroid product development helped establish Syntex and later played a role in the founding of ALZA Corporation.     

Protection against SIV infection in macaques by immunization with inactivated virus from the BK28 molecular clone, but not with BK28-derived recombinant env and gag proteins.

Vaccination of cynomolgus macaques with beta-propiolactone inactivated SIVmacBK28 in Freund's adjuvant induced low but detectable levels of anti-SIV envelope (env) antibodies and T-cell responses and protected against challenge with the 32H isolate of SIVmac251 grown in C8166 cells. In contrast, purified recombinant SIV env and gag proteins derived from BK28 formulated in Syntex adjuvant generated consistent and long-lived cellular and humoral immune responses to SIV env, but failed to protect against infection with the 32H virus. Thus, protection against a heterogeneous challenge stock is possible by immunization with a molecularly-cloned virus, but not with recombinant proteins from the same molecular origin. High levels of anti-cell antibodies induced by the whole virus vaccine, but not by recombinant proteins, may have contributed to the protection observed.     

Spatial structure of the basic chain of the neurotoxin I molecule from the venom of cobra Naja naja oxiana and its crystal packing

The structure of the alpha-carbon chain was solved by molecular replacement method at 2.7 A resolution. Neurotoxin I (NTX-I) is one of the main protein components purified from the venom of the central asian cobra Naja naja oxiana. NTX-I is known to bind specifically to acetylcholine receptors thus preventing the transmission of the neuroconductivity signal from synapses to muscles. NTX-I crystals were grown either by vapour diffusion or dialysis methods using specially prepared microdialysis cell. The intensities of reflections from native NTX-I crystals were measured in the range of 38.0-2.1 A-1 by omega-scan method with a Syntex P21 diffractometer operated in automatic regime. To determine the position and mode of packing of NTX-I molecule in unit cell program packages MERLOT and BLANC were applied running on a NORD-500 computer.     

Induction of luteolysis in the mare with a prostaglandin analogue

Five mares were administered 0.5 to 2.0 mg of a prostaglandin analogue, RS 9390 (Syntex), during nine estrous cycles in February and March. Luteolysis as measured by peripheral plasma progesterone occurred in four cycles, transitory luteolysis following 0.5 mg RS 9390 in two cycles, while functional corpora lutea were not present in three cycles. In 8 out of 9 of these cycles the mares returned to estrus 1.5 to 5 days following treatment. It appears that RS 9390 can be used as a regulator of cycle length in mares.     

Differential inhibition of human placental prostaglandin release in vitro by a GnRH antagonist

Previously, we have demonstrated that the production of prostaglandins by human placental tissue varied with gestational age. In addition, we have shown that placental prostaglandin release was affected by GnRH, and that its response was also dependent on the gestational age of the placenta. Thus, we have studied the effect of a GnRH antagonist ([N-Ac-Pro1,D-p-Cl-Phe2,D-Nal(2)3,6-LHRH, Syntex Research, Palo Alto, CA) on basal prostaglandin release from placentas of 6 to 15 weeks' gestation and found that this antagonist (1 microgram/ml) effects an inhibition of the release of prostaglandin E, prostaglandin F, and 13,14-dihydro-15-keto-prostaglandin from placentas of 13 and 15 weeks of gestation. This effect was not overridden by GnRH at 10 times the antagonist concentration in the 13-week placental cultures, but was totally reversed by GnRH (10 micrograms/ml) in the 15-week placental cultures. These data demonstrate that this GnRH antagonist can affect human placental prostaglandin production at 13 to 15 weeks of gestation and indicate that endogenous placental GnRH-like activity may exert a control over placental prostaglandin release at this gestational stage.     

Differential inhibition of human prostaglandin release in vitro by a GnRH antagonist

Previously, we have demonstrated that the production of prostaglandins by human placental tissue varied with gestational age. In addition, we have shown that placental prostaglandin release was affected by GnRH, and that its response was also dependent on the gestational age of the placenta. Thus, we have studied the effect of a GnRH antagonist ([N-Ac-Pro¹, D-p-Cl-Phe², D-Nal (2)^3,6-LHRH, Syntex Research, Palo Alto, CA) on basal prostaglandin release from placentas of 6 to 15 weeks' gestaton and found that this antagonist (1 μg/ml) effects an inhibition of the release of prostaglandin E, prostaglandin F, and 13, 14-dihydro-15-keto-prostaglandin from placentas of 13 and 15 weeks of gestation. This effect was not overridden by GnRH at 10 times the antagonist concentration in the 13-week placental cultures, but was totally reversed by GnRH (10 μg/ml) in the 15-week placental cultures. These data demonstrate that this GnRH antagonist can affect human placental prostaglandin production at 13 to 15 weeks of gestation and indicate that endogenous placental GnRH-like activity may exert a control over placental prostaglandin release at this gestational stage.     

Transfer and incorporation of genes controlling beta-D-galactosidase synthesis from Hfr and F' donors of Escherichia coli

Ganesan, Ann K. (Syntex Institute of Molecular Biology, Palo Alto, Calif.), and Boris Rotman. Transfer and incorporation of genes controlling beta-d-galactosidase synthesis from Hfr and F' donors of Escherichia coli. J. Bacteriol. 92:1378-1382. 1966.-Comparisons were made between Hfr(1) and F(13) donors with respect to the frequency of transfer and incorporation of genes controlling beta-d-galactosidase synthesis. The Hfr(1) donor transfers these genes as part of the chromosome, and the F(13) donor transfers them by F-duction. The criterion used for gene transfer was the acquisition by recipient cells of the ability to synthesize the enzyme, beta-d-galactosidase, measured by fluorogenic assays at the single-cell level. The criterion for incorporation was the formation of lac(+) recombinant colonies. It was found that the two types of donor showed the same frequency of gene transfer, but the probability of incorporation was 10-fold higher in F(13) matings than in Hfr(1) matings. In the former, between 46 and 97% of the merozygotes produced recombinant colonies; in the latter, 2 to 6% did so.     

Effect of melatonin implants on spermatogenesis in the domestic cat (Felis silvestris catus)

The aim of this study was to assess the efficacy of subcutaneous melatonin implants to temporarily and reversibly suppress spermatogenesis in male cats. Tomcats (n = 8) were housed in a conditioned room with alternating long and short 2-month photoperiod cycles to maintain sperm production and quality. Animals were randomly assigned to one of the two treatments. Four animals received a subcutaneous melatonin implant (MEL, 18 mg; Syntex, Argentina), whereas the other four received a subcutaneous placebo implant (PLA, 0 mg; Syntex). Semen samples were collected by electroejaculation every 14 days for 252 days. Sperm parameters were evaluated in all ejaculates, and data were analyzed by ANOVA. Melatonin-implanted cats significantly decreased their sperm quality in all the parameters studied compared with the control group (MEL vs. PLA; least squares means ± SEM; motility, 71.3 ± 3.4 vs. 82.1 ± 3.6; velocity, 3.4 ± 0.1 vs. 4.6 ± 0.1; total sperm count, 2.6 ± 2.2 vs. 19.4 ± 3.3; acrosome integrity, 48.7 ± 5.6 vs. 62.8 ± 5.6; plasma membrane integrity, 52.2 ± 4.7 vs. 72.9 ± 5.5; normal sperm morphology, 45.8 ± 3.3 vs. 63.7 ± 3.4; P < 0.05). Conversely, volume and serum testosterone concentrations were similar in both groups (volume, 0.15 ± 0.02; serum testosterone concentrations, 1.1 ± 0.1; CV 18.9%; P > 0.05). At 91 ± 7 days after implant insertion, sperm motility decreased 38.5%, velocity 26.5%, total sperm count 82%, acrosome integrity 22%, plasma membrane integrity 30%, and normal sperm morphology decreased 32% of preimplant values. This effect was present until 120 ± 15 days after implant insertion. After that, seminal parameters started to increase and reached preimplant values at about 140 ± 7 days after implant insertion. Nevertheless, treated animals conserved the capacity to produce semen during the treatment period. In conclusion, a single subcutaneous melatonin implant effectively and reversibly reduced sperm production and quality in male domestic cats for approximately 120 ± 15 days without clinically detectable adverse effects.     

An adenovirus-simian immunodeficiency virus env vaccine elicits humoral, cellular, and mucosal immune responses in rhesus macaques and decreases viral burden following vaginal challenge.

Six female rhesus macaques were immunized orally and intranasally at 0 weeks and intratracheally at 12 weeks with an adenovirus type 5 host range mutant (Ad5hr)-simian immunodeficiency virus SIVsm env recombinant and at 24 and 36 weeks with native SIVmac251 gp120 in Syntex adjuvant. Four macaques received the Ad5hr vector and adjuvant alone; two additional controls were naive. In vivo replication of the Ad5hr wild-type and recombinant vectors occurred with detection of Ad5 DNA in stool samples and/or nasal secretions in all macaques and increases in Ad5 neutralizing antibody in 9 of 10 macaques following Ad administrations. SIV-specific neutralizing antibodies appeared after the second recombinant immunization and rose to titers > 10,000 following the second subunit boost. Immunoglobulin G (IgG) and IgA antibodies able to bind gp120 developed in nasal and rectal secretions, and SIV-specific IgGs were also observed in vaginal secretions and saliva. T-cell proliferative responses to SIV gp140 and T-helper epitopes were sporadically detected in all immunized macaques. Following vaginal challenge with SIVmac251, transient or persistent infection resulted in both immunized and control monkeys. The mean viral burden in persistently infected immunized macaques was significantly decreased in the primary infection period compared to that of control macaques. These results establish in vivo use of the Ad5hr vector, which overcomes the host range restriction of human Ads for rhesus macaques, thereby providing a new model for evaluation of Ad-based vaccines. In addition, they show that a vaccine regimen using the Ad5hr-SIV env recombinant and gp120 subunit induces strong humoral, cellular, and mucosal immunity in rhesus macaques. The reduced viral burden achieved solely with an env-based vaccine supports further development of Ad-based vaccines comprising additional viral components for immune therapy and AIDS vaccine development.     

Chinchilla "big" and "little" gastrins

Gastrin heptadecapeptides (gastrins I and II which differ in the presence of sulfate on the tyrosine of the latter) have been purified and sequenced from several mammalian species including pig, dog, cat, sheep, cow, human and rat. A 34 amino acid precursor ("big" gastrin), generally accounting for only 5% of total gastrin immunoreactivity, has been purified and sequenced only from the pig, human, dog and goat. Recently we have demonstrated that guinea pig (GP) "little" gastrin is a hexadecapeptide due to a deletion of a glutamic acid in the region 6-9 from its NH2-terminus and that GP "big" gastrin is a 33 amino acid peptide. The chinchilla, like the GP, is a New World hystricomorph. This report describes the extraction and purification of "little" and "big" gastrins from 31 chinchilla antra. Chinchilla "little" gastrin is a hexadecapeptide with a sequence identical to that of the GP and its "big" gastrin is a 33 amino acid peptide with the following sequence: (See text)     

Opossum (Didelphis virginiana) "little" and "big" gastrins

1. "Little" gastrins from most mammalian species are 17 amino acid peptides and the precursor "big" gastrins are 34 amino acid peptides. 2. "Little" gastrins of the New World hystricomorphs, guinea-pig and chinchilla, are 16 amino acid peptides due to deletion of a glutamic acid in the region 6-9 from their NH2-terminus and the corresponding "big" gastrins are 33 amino acid peptides. 3. Antral gastrins from the opossum, a New World marsupial, have a glutamic acid deletion in the same region as the hystricomorph gastrins. 4. Opossum "big" gastrin is a 33 amino acid peptide with the following sequence: less than ELGPQDLPYLTADLSKKQGPWLEEEEAYGWMDF#.     

Cognizing "Cognized Models"

In his later work Rappaport acknowledged that his earlier efforts had overemphasized organic and ecological functions in the explanation of cultural phenomena. He then distanced himself from both idealism and reductive materialism and set out to understand the complexities of cultural understandings and ritual. Specifically, he shifted from functionalism to formalism in an effort to understand ritual and its language in relation to cultural norms. Ultimately his analysis was implicitly structural, understanding the part as a constituent of an overarching arrangement and in terms of what Althusser would have called "structural causality." Although his work benefited from this shift from function to structure, Rappaport did not use it to explore the political dimension. However, a holistic ecology such as the one Rappaport essayed must ultimately embrace both political ecology and historical ecology, [cognized models, ritual, political ecology, structural causality-]     

cAMP regulation of "pathogenic" and "saprophytic" fungal spore germination

We report on the elucidation of two separate pathways of spore germination in a plant pathogenic fungus Colletotrichum gloeosporioides f. sp. aeschynomene. Conidia of the fungus can germinate either from one side or from both sides, depending on external conditions. In shake culture that includes an extract made up from fresh peas, the unicellular conidium divides and one of the two cells develops a germ tube. On a solid surface this germ tube differentiates an appressorium. In rich medium without pea extract, germination is highly similar to Aspergillus spore germination: the conidium swells, forms a single germ tube and then divides and forms a second germ tube. Conidia that germinate in a rich medium do not form appressoria even on a solid surface and are non-pathogenic. In rich medium, cAMP stimulates germination in rich liquid cultures and induces appressoria formation on a hard surface. In pea extract cAMP induces swelling and formation of irregular germ tubes and appressoria. Our results suggest that plant surface signals induce pathogenic-specific spore germination in a cAMP-independent manner. cAMP is required for saprophytic germination and for appressorium formation.     

蕲州地区的蕲艾、青蒿、黄花蒿与茵陈的考订

<正> 蕲州是我国中药材主要产地之一,是湖北省蕲春县的一个集镇,,是我国中医药大师李时珍的家乡。该地区生长的菊科蒿属植物Artemisia Linn。颇多,其中入药的有十余种,并在李时珍《本草纲目》中曾有记载。这里仅对该地区常见入药的蕲艾、青蒿、黄花蒿与茵陈结合《本草纲目》记载的材料作初步的考订。     

"Light" and "dark" pinealocytes of the porcine pineal gland

Both qualitative and quantitative comparative studies of "dark" and "light" pinealocytes of the porcine pineal gland have been carried out. These cells differ from each other in their electronic density of cytoplasm, shape of nucleus, the structure of membrane bound dense bodies and the number of microtubules and smooth endoplasmic reticulum. The membrane bound dense bodies--characteristic structures of pig pinealocytes as well dense core vesicles occur in both types of cells. The relative volume of the majority of the cells' organellae apart from the Golgi apparatus, also do not show any significant difference. The results obtained support a functional basis for pinealocyte differentiation in the porcine pineal gland.     

On the controversy over "fast" and "slow" helix-coil transition rates in polypeptides

The controversy over “fast” and “slow” helix-coil transition rates in polypeptides is discussed. The “slow” results are derived from the assumption that multiple NMR spectra of α-CH and NH groups arise from chemical exchange. In this paper it is shown that such spectra may be obtained without invoking chemical exchange. The multiplicity arises from the difference in helicity of amino acid residues near the ends of the chain by comparison with amino acid residues nearer the middle, and from a polydispersity in molecular weight. As a consequence of this analysis, support is given to the “fast” transition rates.     

High-resolution 2-dimensional protein mapping of "diploid" and "aneuploid" mammary adenocarcinomas

Two-dimensional-electrophoretic analysis has been applied to non-neoplastic mammary epithelium from eight healthy women, tumor tissue from eight "diploid" mammary adenocarcinomas, and tumor tissue from eight "aneuploid" mammary adenocarcinomas. Compared with non-neoplastic mammary epithelium, a slight numerical net non-neoplastic mammary epithelium, a slight numerical net increase of the protein spots was detected in "diploid" tumors and a marked increase in "aneuploid" tumors. Two prominent spots were present in all 16 malignant tissues examined and absent in all eight non-neoplastic tissues (silver staining method). The results suggest that a difference in the composition of cellular proteins exists both between non-neoplastic mammary cells and malignant tumor cells, and between "diploid" and "aneuploid" tumors.     

Fracture faces of zonulae occludentes from "tight" and "leaky" epithelia

Epithelia vary with respect to transepithelial permeability. In those that are considered "leaky", a large fraction of the passive transepithelial flux appears to follow the paracellular route, passing across the zonulae occludentes and moving down the intercellular clefts. In "tight" epithelia, the resistance of the paracellular pathway to passive flux is greatly increased. To see whether differences in the morphology of the zonula occludens could contribute to this variability in leakiness among epithelia, replicas of zonulae occludentes in freeze-fractured material from a variety of tight and leaky epithelia were examined. The junctions appear as a branching and anastomosing network of strands or grooves on the A and B membrane fracture faces, respectively. It was found that the zonula occludens from a "very leaky" epithelium, the proximal convoluted tubule of the mouse kidney, is extremely shallow in the apical-basal direction, consisting in most places of only one junctional strand. In contrast, the "very tight" frog urinary bladder exhibits a zonula occludens that is relatively deep (>0.5 µm) in the apical-basal direction, and consists of five or more interconnected junctional strands interposed between luminal and lateral membrane surfaces. Epithelia of intermediate permeabilities exhibited junctions with intermediate or variable morphology. Toad urinary bladder, mouse stomach, jejunum, and distal tubule, rabbit gallbladder, and Necturus kidney and gallbladder were also examined, and the morphological data from these epithelia were compared to physiological data from the literature.     

Physical separation of "suppressor" from "helper" thymocytes.

Thymocytes from 1-month-old NZB/W mice were separated at unit gravity and then studied for helper or suppressor effects in a GVH assay. Thymocytes with a settling rate of 4 to 6 mm/hr suppressed but did not help. In contrast, help but no suppression characterized thymocytes settling at 6 to 10 mm/hr. Thus helper and suppressor thymocytes in this GVH assay were physically separated.