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Antiserum sheep 3 against rat brain glutamate decarboxylase (GAD) was employed for the immunohistochemical localization of GAD-immunoreactive nerve terminals in the substantia nigra (SN). To test whether the antiserum specifically localized GAD-containing axon terminals, the effect of kainic acid-induced striatal lesions on the reactive nerve endings in the SN was investigated. Seven days after the injection of 1 microgram kainic acid into the striatum, a 65% decrease in GAD-enzyme activity occurred in the ipsilateral SN. On immunohistochemical examination there was correspondingly a marked reduction of GAD-positive terminals. The parallel decrease in biochemical and immunohistochemical GAD activity indicates that antiserum sheep 3 can be used as a specific immunohistochemical probed for GAD-containing elements.

The cellular localization of neurons expressing cholecystokinin (CCK) and tyrosine hydroxylase (TH) mRNAs was analysed in rat ventral mesencephalon using in situ hybridization techniques with both complementary DNA and synthetic oligonucleotide probes. Cell bodies distributed throughout the substantia nigra, ventral tegmental area, interfascicular nucleus, midline raphe nuclei, and central and ventral periaqueductal grey matter were found to contain CCK mRNA or TH mRNA as indicated by high densities of grains overlying the perikarya. The in situ hybridization technique was combined with immunocytochemistry on the same tissue section to localize the peptide or enzyme within its respective mRNA-containing somata. In addition, the presence of TH immunoreactivity was demonstrated within cell bodies labeled for CCK mRNA and immunostaining for CCK was detected within TH mRNA-containing neurons. In the medial geniculate nucleus a strong labeling for CCKmRNA was observed, in spite of the fact that so far no CCK-like immunoreactivity has been demonstrated in perikarya in this nucleus. The specificity of the probes was verified by RNA blot hybridization. These results confirm recent double-labeling immunocytochemical studies and further characterize the coexistence of CCK and TH at the level of their mRNAs as well as their post-translational products in a large population of mesencephalic dopamine neurons known to project to forebrain areas.

Large-scale genotyping is required to generate dense identity-by-descent maps to map genes for human complex disease. In some studies the number of genotypes needed can approach or even exceed 1 million. Generally, linkage and linkage disequilibrium analyses depend on clear allele identification and subsequent allele frequency estimation. Accurate grouping or categorization of each allele in the sample (allele calling or binning) is therefore an absolute requirement. Hence, a genotyping system that can reliably achieve this is necessary. In the case of affected sib-pair analysis without parents, the need for accurate allele calling is even more critical. We describe methods that permit precise sizing of alleles across multiple gels using the fluorescence-based, Applied Biosystems (ABI) genotyping technology and discuss ways to reduce genotyping error rates. Using database utilities, we show how to minimize intergel allele size variation, to combine data effectively from different models of ABI sequencing machines, and automatically bin alleles. The final data can then be converted into a format ready for analysis by statistical genetic packages such as MENDEL.

[Arg8]vasopressin (AVP) stimulates adrenocorticotropic hormone release from the anterior pituitary by acting on the V1b AVP receptor. This receptor can be distinguished from the vascular/hepatic V1a and renal V2 AVP receptors by its differential binding affinities for structural analogous of AVP. Recent studies have shown that the cloned V1a and V2 receptors are structurally related. We have isolated a clone encoding the V1b receptor from a rat pituitary cDNA library using polymerase chain reaction (PCR)-based methodology. The rat V1b receptor is a protein of 421 amino acids that has 37-50% identity with the V1a and V2 receptors. Homology is particularly high in the seven putative membrane-spanning domains of these guanine nucleotide-binding protein-coupled receptors. Expression of the recombinant receptor in mammalian cells shows the same binding specificity for AVP agonists and antagonists as the rat pituitary V1b receptor. AVP-stimulated phosphotidylinositol hydrolysis and intracellular Ca2+ mobilization in Chinese hamster ovary or COS-7 cells expressing the cloned receptor suggest second messenger signaling through phospholipase C. RNA blot analysis, reverse transcription PCR, and in situ hybridization studies reveal that V1b receptor mRNA is expressed in the majority of pituitary corticotropes as well as in multiple brain regions and a number of peripheral tissues, including kidney, thymus, heart, lung, spleen, uterus, and breast. Thus, the V1b receptor must mediate some of the diverse biological effects of AVP in the pituitary as well as other organs.

Sensitive radioisotopic enzymatic methods were used to determine 5-HT levels in 16 different regions of brain from rats anesthetized for 90–105 min with 1% halothane or 18% cyclopropane. These two anesthetics were chosen because of their differing effects on the electroencephalogram and on the cardio-vascular and respiratory systems. 5-HT levels in the nucleus amygdaloideus centralis, substantia nigra, and nucleus centralis superior were increased after administration of either anesthetic, but only anesthesia with cyclopropane was associated with an increase in 5-HT level in the nucleus raphe dorsalis. The changes in levels of transmitter does not distinguish cause from effect of anesthesia, and further experiments are needed to delineate what role, if any, the specific areas play in muscle relaxation, analgesia, sleep or anesthesia.

Large-scale genotyping is required to generate dense identity-by-descent maps to map genes for human complex disease. In some studies the number of genotypes needed can approach or even exceed 1 million. Generally, linkage and linkage disequilibrium analyses depend on clear allele identification and subsequent allele frequency estimation. Accurate grouping or categorization of each allele in the sample (allele calling or binning) is therefore an absolute requirement. Hence, a genotyping system that can reliably achieve this is necessary. In the case of affected sib-pair analysis without parents, the need for accurate allele calling is even more critical. We describe methods that permit precise sizing of alleles across multiple gels using the fluorescence-based, Applied Biosystems (ABI) genotyping technology and discuss ways to reduce genotyping error rates. Using database utilities, we show how to minimize intergel allele size variation, to combine data effectively from different models of ABI sequencing machines, and automatically bin alleles. The final data can then be converted into a format ready for analysis by statistical genetic packages such as MENDEL.

Specific rabbit antisera against purified bovine pituitary carboxypeptidase processing enzyme (which has also been referred to as "enkephalin convertase") have been prepared and characterized. The antisera recognized both the purified soluble and the membrane-bound forms of the enzyme with equal affinity, suggesting that these two forms of the enzyme may possess many regions of structural homology. Since the antisera did not crossreact with carboxypeptidases B, N, A, Y, and P, the carboxypeptidase processing enzyme may be a structurally distinct form of carboxypeptidase. Carboxypeptidase immunostaining, as seen by light microscopy, was found throughout the rat brain and in bovine adrenal medulla, reflecting the widespread distribution of neuropeptides. Electron microscopic immunocytochemistry of rat paraventricular nucleus and other brain areas showed that the enzyme was present in some dendrites and nerve terminals, which contain storage vesicles. These findings support the hypothesis that this carboxypeptidase is involved in the processing of many peptide hormone precursors.

Carboxypeptidase H is a putative post-translational processing enzyme which removes basic amino acid residues from intermediates during protein hormone biosynthesis. A 2.2-kilobase pair cDNA was shown to contain the complete amino acid sequence of rat carboxypeptidase H. The deduced amino acid sequence revealed that the enzyme was synthesized as preprocarboxypeptidase H, a precursor form of 476 amino acid residues. Preprocarboxypeptidase H contained a putative hydrophobic signal peptide and a short propeptide which contained 5 adjacent Arg residues at its C terminus. Northern blot analysis identified a single carboxypeptidase H mRNA of ∼2.3 kilobases in brain, pituitary, and heart, as well as in mouse AtT20 cells. No carboxypeptidase H mRNA was detected in rat liver, spleen, kidney, lung, and mammary gland. Sequence analysis of cDNAs obtained from different rat tissues suggested that a single mRNA encodes an identical carboxypeptidase in several tissues. Treatment of AtT20 cells with dexamethasone decreased the levels of both carboxypeptidase H and preproopiomelanocortin (POMC) mRNAs by ∼30%. Exposure of the dexamethasone-treated cells to corticotropin-releasing factor effected a 2- to 3-fold increase in the carboxypeptidase H and POMC mRNA levels relative to those of dexamethasone-treated cells exposed to control medium. This suggests that the mRNA levels of POMC and one of its putative post-translational processing enzymes, carboxypeptidase H, are co-regulated by corticotropin-releasing factor and steroid hormones.

The primary structure of gonadotropin-releasing hormone (GnRH) isolated from whole brains of lamprey is pGlu-His-Tyr-Ser-Leu-Glu-Trp-Lys-Pro-Gly-NH2. This unique decapeptide was isolated and purified from brain extracts by reverse-phase high performance liquid chromatography. The structure of the peptide was established from chymotryptic fragments that were identified by protein sequence analysis and fast atom bombardment mass spectrometry. The peptide reacts with an antiserum raised against mammalian GnRH and is structurally identified as a member of the GnRH family by the amino and carboxyl termini of pGlu1-His2 and Pro9-Gly10NH2, the conservation of Ser4 in the internal segment of the molecule and its length of 10 amino acids. For the first time, amino acid substitutions are found in positions 3 and 6, critical for biological potency and conformation, respectively. Additionally, a second form of GnRH (lamprey II GnRH), representing about 10% of the total GnRH immunoreactive material in the brain, was isolated; its amino acid composition differs by 3 residues from lamprey I GnRH. Synthetic lamprey I GnRH elevates plasma estradiol in adult female lampreys.

Prodynorphin-derived peptides [dynorphin A (Dyn A)-(1-17), Dyn A-(1-8), Dyn B, alpha-neo-endorphin, and beta-neo-endorphin] and proenkephalin-derived peptides [[Leu]enkephalin [( Leu]Enk) and [Met]enkephalin-Arg6-Gly7-Leu8 [( Met]Enk-Arg-Gly-Leu]) in selected brain areas of the rat were measured by specific radioimmunoassays. We report here that different regions of rat brain contain strikingly different proportions of the prodynorphin and proenkephalin-derived peptides. There is a molar excess of alpha-neo-endorphin-derived peptides over Dyn B and Dyn A-derived peptides in many brain areas. [Leu]Enk concentrations exceed those of [Met]Enk-Arg-Gly-Leu in certain brain areas such as the substantia nigra, dentate gyrus, globus pallidus, and median eminence (areas rich in dynorphin-related peptides). These results indicated that (i) there is differential processing of prodynorphin in different brain regions and (ii) [Leu]Enk may be derived from Dyn A or Dyn B (or both). In certain brain regions [Leu]Enk may derive from two separate precursors (prodynorphin and proenkephalin) in two distinct neuronal systems.

Immunoreactive and bioreactive corticotropin (ACTH-like) activities have been detected in the median eminence and remaining medial basal hypothalamus of both normal and hypophysectomized adult male rats: bioreactive ACTH (pg/100 mug of protein) 1028 in median eminence and 1289 in medial basal hypothalamus; immunoreactive ACTH (midportion ACTH antibody), 1554 in median eminence and 1887 in medial basal hypothalamus. By use of appropriate antibodies and bioassay, it was demonstrated that immunoreactivity was not due solely to alpha-melanotropin, which has previously been reported to be present in the brain of hypophysectomized animals. The Sephadex G-50 gel filtration patterns determined by immunoassay of column eluates obtained from hypothalamic extracts of normal or hypophysectomized animals were similar but were not identical to the pattern derived from whole pituitary. Immunoreactive (midportion ACTH antibody) ACTH concentrations (pg/100 mug of protein) of other central nervous system areas in normal animals were: cerebellum 34.3, cortex 46.3, thalamus 23.8, and hippocampus 116.3. The total amount of bioreactive ACTH present in the median eminence and medial basal hypothalamus is approximately 1% of that present in the pituitary. The present data suggest that such ACTH may have a diencephalic rather than pituitary origin and raise the question of the functional significance of such ACTH.

When [35S]cysteine was injected adjacent to the supraoptic nucleus (SON) in rats, it was rapidly incorporated into proteins in the SON. The [35S]cysteine-labeled proteins extracted from the SON were separated by isoelectric focusing on polyacrylamide gels. Twenty minutes after the injection of [35S]cysteine, two major labeled peaks (pI = 5.4 and 6.1) were found in the SON of normal rats; Brattleboro rats had only one major labeled peak (pI = 5.4). One hour after the injection, four major radioactive peaks were found in the SON of normal animals (pI = 5.1, 5.4, 5.6, and 6.1). Animals with diabetes insipidus had only two major labeled proteins (pI = 5.1 AND 5.4). Twenty-four hours after normal rats were injected with [35S]cysteine, all of the labeled peaks described above, except for the one with pI = 5.1, had decreased markedly in size and a small amount of labeled protein with pI about 4.8 was present in the SON. After 24 hr the posterior pituitary of normal animals contained two [35S]cysteine-labeled proteins with pI = 4.6 AND 4.8. The pituitaries of Brattleboro rats had only the pI = 4.6 labeled protein. These pulse-chase data, with data we have presented elsewhere, indicate that the vasopressin- and oxytocin-neurophysins are synthesized as parts of separate precursors (pI = 6.1 and 5.4, respectively). These precursors are converted into at least two intermediates (pI = 5.6 and 5.1) which, in turn, yield the vasopressin-neurophysin (pI = 4.8) and the oxytocin-neurophysin (pI = 4.6).

Insulin concentrations in acid/ethanol extracts of the whole rat brain were on the average 25 times higher than plasma insulin levels. Brain insulin was indistinguishable from authentic pancreatic insulin, based on its behavior in radioimmunoassay, radioreceptor assay, and bioassay and its chromatographic pattern on Sephadex G-50 column chromatography. Insulin was found in all regions of the brain examined, but distribution was uneven. Some regions had insulin concentrations as much as 100 times higher than in plasma; levels at least 10 times higher were found in other regions. The role of insulin in the central nervous system is not clear at present but, because both insulin and insulin receptors are abundant in the central nervous system, an extensive physiological regulation of the central nervous system by insulin is proposed.

Although the hypothesis that vasopressin and its associated neurophysin are synthesized together in one macromolecular common precursor was put forward more than a decade ago, direct conformation of this hypothesis has been lacking. A [35S]cysteine-labeled putative precursor for vasopressin-related neurophysin (Mr 20,000, pI 6.1) has been isolated from the supraoptic nuclei of rats. This precursor was subjected to limited proteolysis with trypsin which produced a Mr 10,000 protein and peptide products. The former was identified as neurophysin on the basis of its pH-dependent affinity for vasopressin and its behavior in isoelectric focusing systems (pI 4.6-4.8). The tryptic peptides proved to be vasopressin-like because they: (i) were rich in cysteine, (ii) comigrated with vasopressin on gel filtration columns in 6 M guanidine HCl, (iii) bound to a neurophysin-Sepharose affinity column at pH 5.7, and (iv) were recognized by antibodies against vasopressin. These data on the Mr 20,000, pI 6.1 protein represent direct experimental evidence for a candidate for the common precursor of vasopressin and neurophysin. We propose that this common precursor be called "propressophysin."

Background.Lack of skin for autograft continues to be problematic in patients with large burns. Allograft and xenograft have been used, but are prone to rapid rejection. Use of cultured keratinocytes (CK) and major histocompatibility complex (MHC) II “knockout” grafts leads to prolonged graft survival compared to allograft. Whether this prolongation is secondary to decreased priming efficacy or target recognition is unknown. Whether a combination of these techniques would generate a less immunogenic allograft remains to be determined.Methods.CBA mice (n= 100) were flank-grafted with full thickness C57BL/6 (B6 FT), B6 cultured keratinocytes (B6 CK), B6 major histocompatibility complex II “knockout” full thickness (KO II FT), B6 major histocompatibility complex II “knockout” cultured keratinocytes (KO II CK), or a full thickness autograft (Auto). Three weeks after priming flank grafting, B6, MHC I (KO I), and KO II full thickness tail grafts were placed on each mouse. Tail graft rejection was assessed daily by an observer blinded to flank and tailgraft type. A 4-point grading system for graft color, hair loss, and texture was used.Results.Animals primed with KO II CK flank grafts had increased survival of tail grafts over B6 FT flank grafted controls (12.3 ± 1.05 vs 10.1 ± 1.00,P< 0.05). Within flank graft groups, however, B6, KO I, and KO II tail graft survival was similar.Conclusions.KO II CK allografts decrease host priming compared to normal B6 FT allograft. MHC deletion (KO I or KO II) does not protect a target graft from rejection in a primed host. CK and KO techniques may offer a less immunogenic allograft and a readily available source of wound coverage in patients with extensive burns.

The presence of the 29-amino acid peptide galanin and galanin-like immunoreactivity in the peripheral and central nervous system have been established. We describe below the construction of a 1.1-million-recombinant cDNA library derived from poly(A)+ RNA from pig adrenal medulla, rich in galanin-like immunoreactivity, and the isolation and characterization of clones encoding preprogalanin. Mixtures of synthetic oligodeoxyribonucleotides corresponding to galanin-(7-19) and galanin-(22-29) were synthesized and used as hybridization probes to screen the cDNA library. Sequencing of two recombinant plasmids shows that pig galanin mRNA in the adrenal medulla encodes a precursor protein of 123 amino acids that is comprised of a leader sequence, a single galanin sequence, and a 59-amino acid sequence (galanin message-associated peptide) of as yet unknown function. The single galanin sequence, followed by a glycine (the amide donor), is flanked on both sides by pairs of lysine and arginine residues. RNA blot-hybridization analysis of poly(A)+ RNA from pig adrenal medulla reveals a single mRNA of approximately equal to 900-950 bases.

Somatostatin-like activity, as determined by radioimmunoassay and bioassay, is present in HCl extracts of rat retina. The concentrations of immunoreactive somatostatin are 612 +/- 43 (mean +/- SEM)pg per whole retina of 0.621 +/- 0.044 pg/microgram of protein in retinas from rats killed by decapitation, values which are not significantly different from those in retinas from rats killed by ether inhalation. The immunoreactive somatostatin was partially purified and concentrated by immunoaffinity chromatography. Both the crude retinal extracts and the immunoaffinity-purified immunoreactive somatostatin inhibited, in a dose-related manner, the release of rat growth hormone from dispersed rat anterior pituitary cells in culture. When the immunoaffinity-purified immunoreactive somotostatin was subjected to gel filtration chromatography, 96% of the recovered somatostatin eluted as a peak corresponding in position to that of synthetic somatostatin. Retinas from a group of rats with hereditary degeneration of the photoreceptor cells and another group of rats studied 1 year after transection of the optic nerves demonstrated an increased concentration of immunoreactive somatostatin compared to controls.

Insulin has been detected, at levels higher than those in plasma, in a broad range of extrapancreatic tissues in both rats and humans. Rat liver insulin was shown to be indistinguishable from genuine insulin by radioimmunoassay, Sephadex chromatography, bioassay, and antibody neutralization. Liver insulin (like brain insulin) was unchanged in ob/ob mice, in rats treated with streptozotocin, or in fasted rats, despite marked alterations in pancreatic secretion of insulin and in liver content of insulin receptors. Insulin was found in cultured human IM-9 lymphocytes and cultured fibroblasts at concentrations greater than 100 times the levels in the media. IM-9 lymphocyte insulin also was shown to be indistinguishable from genuine insulin, by the same criteria used for liver insulin. The insulin concentration in cultured human cells was unaffected by depletion of insulin from the culture medium or by addition of beef insulin to the medium. The data suggest that a part, if not all, of the extrapancreatic tissue insulin is independent of plasma insulin and may be synthesized by the tissues themselves.

A peptide that is recognized by certain antibodies raised against mammalian gonadotropin-releasing hormone has been purified from extracts of salmon brains by gel filtration and high-performance liquid chromatography. The primary structure of this 10-residue peptide is less than Glu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH2. This represents a difference of two amino acids between salmon and mammalian gonadotropin-releasing hormone and demonstrates that most of the molecule has been conserved during evolution. The synthetic form of salmon gonadotropin-releasing hormone is less potent than is mammalian gonadotropin-releasing hormone on mammalian cells and is biologically active in salmon.

Previous work in our laboratory has shown that stimulation of beta 2-adrenergic receptors on mouse anterior pituitary tumor cells causes the secretion of immunoreactive adrenocorticotropin (ACTH). The present study was designed to test the hypothesis that catecholamines can cause the release of ACTH in vivo by the direct stimulation of beta 2-adrenergic receptors in the rat anterior pituitary. Systemic administration of a beta-adrenergic receptor agonist (-)-isoproterenol resulted in an increase in plasma ACTH levels in intact animals and in rats with transected pituitary stalks. This effect could be blocked by the beta-adrenergic receptor antagonist, propranolol, but not by the specific beta 1-adrenergic receptor antagonist, practolol. Salmefamol, a beta 2-adrenergic receptor agonist also elevated plasma ACTH levels in stalk-sectioned animals. Dexamethasone, a glucocorticoid that inhibits the synthesis and release of ACTH from the anterior pituitary but not the intermediate lobe, prevented the elevation of ACTH secretion by (-)-isoproterenol in stalk-transected rats. These data indicate that beta 2-adrenergic receptors are present on anterior pituitary cells and suggest that catecholamines can directly stimulate ACTH secretion.

35S-cysteine injected adjacent to the supraoptic nucleus (SON) of the rat is rapidly incorporated into proteins. These 35S-cysteine-labeled proteins in the SON (1-24 h after injection) were separated by polyacrylamide gel electrophoresis, and the distribution of radioactive proteins on the gels was analyzed. 1 h after injection, about 73% of the radioactivity appeared in two peaks (both about 20,000 mol wt). With time, these peaks (putative precursors of neurophysin) decreased, as a 12,000 mol wt peak (containing two distinct neurophysins) increased in radioactivity. Both the 20,000- and 12,000-mol wt proteins are transported into the axonal (median eminence) and nerve terminal (posterior pituitary) regions of the rat hypothalamo-neurohypophysial system. Conversion of the larger precursor protein to the smaller neurophysin appears to occur, in large part, intra-axonally during axonal transport. Six distinct 35S-cysteine-labeled peptides (less than 2500 mol wt), in addition to arginine vasopressin and oxytocin, are also synthesized in the SON and transported to the posterior pituitary where they are released together with labeled neurophysin by potassium depolarization in the presence of extracellular calcium. These data provide support for the hypothesis that the neurohypophysial peptides (vasopressin and oxytocin) and neurophysins are derived from the post-translational clevage of protein precursors synthesized in the SON, and that the conversion process can occur in the neurosecretory granule during axonal transport.

A cDNA encoding the dopamine transporter from bovine brain substantia nigra was identified on the basis of its structural homology to other, recently cloned, neurotransmitter transporters. The sequence of the 693-amino acid protein is quite similar to those of the rat gamma-aminobutyric acid, human norepinephrine, and rat serotonin transporters. Dopamine transporter mRNA was detected by in situ hybridization in the substantia nigra but not in the locus coeruleus, raphe, caudate, or other brain areas. [3H]Dopamine accumulation in tissue culture cells transfected with the cDNA was inhibited by amphetamine, cocaine, and specific inhibitors of dopamine transport, including GBR12909.

Pharmacological and molecular cloning studies have demonstrated heterogeneity of alpha 1-adrenergic receptors. We have now cloned two alpha 1-adrenergic receptors from a rat cerebral cortex cDNA library, using the hamster alpha 1B-adrenergic receptor as a probe. The deduced amino acid sequence of clone RA42 encodes a protein of 560 amino acids whose putative topology is similar to that of the family of G-protein-coupled receptors. The primary structure though most closely resembles that of an alpha 1-adrenergic receptor, having approximately 73% amino acid identity in the putative transmembrane domains with the previously isolated hamster alpha 1B receptor. Analysis of the ligand binding properties of RA42 expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique alpha 1-adrenergic receptor pharmacology. High affinity for the antagonist WB4101 and agonists phenylephrine and methoxamine suggests that cDNA RA42 encodes the alpha 1A receptor subtype. Northern blot analysis of various rat tissues also shows the distribution expected of the alpha 1A receptor subtype with abundant expression in vas deferens followed by hippocampus, cerebral cortex, aorta, brainstem, heart and spleen. The second alpha 1-adrenergic receptor cloned represents the rat homolog of the hamster alpha 1B subtype. Expression of mRNA for this receptor is strongly detected in liver followed by heart, cerebral cortex, brain stem, kidney, lung, and spleen. This study provides definitive evidence for the existence of three alpha 1-adrenergic receptor subtypes.

While insulin effects on the central nervous system (CNS) mediated through hypoglycaemia are well known, direct insulin effects on the CNS remain controversial. Recently, we found insulin receptors in all areas of the rat brain, with highest concentrations in the olfactory bulb, cerebral cortex and hypothalamus; all areas involved in feeding. Insulin receptors in brain were, by multiple criteria, similar to insulin receptors on classical target tissues for insulin, such as liver and fat. Insulin itself has been identified in the rat brain at concentrations on average ten times higher than in plasma. Highest concentrations were found in the olfactory bulb and hypothalamus. Brain insulin was indistinguishable from purified insulin by its behaviour in the radioimmunoassay, radioreceptor assay, bioassay and gel chromatography. In two experimental models representing extremes of plasma insulin concentrations (obese hyperinsulinaemic mice and diabetic insulinopenic rats) there were no significant changes in the concentration of insulin receptors in brain while liver receptors were modified in the expected way. This may reflect the protective influence of the blood-brain barrier or some special quality of brain insulin receptors. Insulin concentrations in brain were also unchanged in both models, which is probably indicative of the local synthesis of insulin. The role of insulin in the CNS is unknown. Besides well known metabolic actions of insulin, new roles can be postulated such as neurotransmission, neuromodulation and paracrine signalling.

The structure of the solid adducts of SF4with several fluoride acceptors has been studied by investigations of the 18F exchange between S18F4and BF3and between SF4and B18F4, the infrared spectrum of SF4.BF3, and the Raman spectra of SF4.BF3, SF4.PF5, SF4.AsF5, and SF4.SbF5. The experimental observations are best accounted for in terms of an ionic structure SF3+.MXn−in each case, but there is good evidence for relatively strong fluorine bridging between the ions.In anhydrous hydrogen fluoride solution, SF4and the adducts SF4.BF3and SF4.SbF5were studied conductimetrically and by 19F nuclear magnetic resonance. Both methods indicate complete ionization of the adducts to SF3+and the complex fluoroanion. SF4is a weak base in hydrogen fluoride ionizing to SF3+and HF2−.

The history of Howard Hughes illustrates an unusual variant or end state of obsessive-compulsive neurosis in which a germ phobia coincides with the deliberate avoidance of cleaning and neglect of personal hygiene. We have reported on three patients who share similar symptomatology. In these cases, the coincidence of personal filthiness and a germ phobia appear paradoxical.

Material reacting with an antibody to thyrotropin-releasing hormone (TRH) has been found to be present in the rat retina. The compound present in the retina cochromatographed with authentic TRH and most of its activity was lost when incubated with pyroglutamate aminopeptidase (L-pyroglutamyl-peptide hydrolase, EC 3.4.11.8), an enzyme that degrades TRH. The TRH-like activity in the rat retina was low during the night and high during the day. There was a 4-hr lag period after the lights were turned on before peak TRH levels were attained. A decrease in TRH was seen after 2 hr of darkness and the level of TRH was lowest after 4 hr of darkness. Retinal TRH is elevated by environmental lighting regardless of the time of the day. These findings suggest that TRH may be involved in retinal photorecptive mechanisms.

The medial basal hypothalamus was isolated from the remainder of the brain of the rat using a Halász knife. Ten to 14 days after the surgical procedure the concentration of thyrotropin-releasing hormone within the island of hypothalamic tissue was 76% lower than in tissue from sham-operated control rats. Thus, much of the thyrotropin-releasing hormone that is normally present in the medial basal hypothalamus may be synthesized by cells outside of this region. There were no reductions in hormone levels in regions outside of the hypothalamic island after the surgical procedure. Extrahypothalamic thyrotropin-releasing hormone does not appear to be produced by hypothalamic neurosecretory cells.