Androgenic control of immunoreactive somatostatin in the Harderian gland of the Syrian hamster

Harderian glands of Syrian hamsters contained measurable levels of immunoreactive somatostatin. After an extraction procedure, serial dilutions of tissue were assayed and showed parallelism in the displacement curve with dilutions of purified somatostatin standard in the radioimmunoassay. Somatostatin concentrations were higher in female hamsters (10.0 +/- 2.1 ng/mg protein) than in males (2.6 +/- 0.4 ng/mg protein). Castrated males had somatostatin values in the range of females (12.4 +/- 2.3 ng/mg protein) at 1 month after gonadectomy. Testosterone implants prevented the rise of Harderian gland somatostatin in castrated males. Gonadectomized males had lower somatostatin content in the gland than did control males (1.0 +/- 0.2 ng/mg protein) at 2 months after castration. Somatostatin values in females were unaffected by gonadectomy, but there were variations during the oestrous cycle, with a nadir detected at dioestrus-1 and maximal values coincident with the day of the ovulation.     

Insulin releasing activity of gastrointestinal glucagon-like immunoreactive materials in perfused rat pancreas

Insulin-releasing activity of porcine gastrointestinal glucagon-like immunoreactive materials purified by affinity chromatography was examined in the perfused rat pancreas. When glucose concentration of the perfusate was raised from 60 to 100 mg/dl, augmented insulin release was observed. The mean incremental area of immunoreactive insulin (sigma delta IRI) during the first 10 min thus observed was 19.07 +/- 3.76 ng/10 min. Pancreatic glucagon and the extract from the gastric fundus showed the enhancement of insulin release in this system when they were added to the perfusate at the rate of 100 ng/min for 5 min; delta IRI were 41.92 +/- 8.47 and 71.70 +/- 18.09 ng/10 min, respectively, which were significantly higher than that of 100 mg/dl of glucose alone. However, no significant difference in the insulinogenic activity was noticed between the extracts from the small intestine and the control. These results suggest that the extract from the gastric fundus has insulinogenic activity similar to that of pancreatic glucagon.     

Lipid metabolism in cultured cells. XVI. Lipoprotein binding and HMG CoA reductase levels in normal and tumor virus-transformed human fibroblasts.

The loss in feedback control of cholesterol biosynthesis in tumor cells was examined in tissue culture. Human fibroblasts from normal subjects, SV40 tumor virus-transformed cell lines, and homozygous familial hypercholesterolemic cells as reference, were grown in tissue culture. Experiments were conducted to relate the regulatory enzyme for cholesterol biosynthesis, HMG CoA reductase, and the membrane-located binding receptors for low density lipoproteins (LDL) that mediate feedback control in normal cells. Monolayers of virus-transformed tumor cells exhibited specific (125)I-labeled LDL binding of 152 +/- 21 ng/mg cell protein, which was essentially the same as that of normal fibroblasts (135 +/- 20 ng/mg). Binding of LDL by familial hypercholesterolemic cells used as controls was only 8 +/- 3 ng/mg under the same test conditions. Basal levels of HMG CoA reductase in tumor cells of 45.2 +/- 6.5 units/mg cell protein were about twice those of normal cells. However, in contrast to the lack of feedback control of this enzyme observed with tumors in vivo, in both the normal and the transformed cells in vitro, activity of the enzyme decreased about fourfold when serum lipids were added. These findings demonstrate that tumor cells growing in vitro contain a normal complement of the membrane-located binding receptors for low density lipoproteins and, although the basal levels are higher than normal, an effective feedback regulation of the enzyme HMG CoA reductase is retained.     

In-vitro venous prostacyclin production,plasma 6-keto-prostaglandin F1 alpha concentrations,and diabetic retinopathy.

Previous studies have shown that vessels from diabetics produce less prostacyclin in vitro than those from normal controls. To determine whether this decreased production is related to complications elective biopsy of a superficial forearm vein was performed on 12 insulin-dependent male diabetics, six with nil or minimal and six with proliferative retinopathy, and seven male controls. Vein segments from the diabetics and controls produced similar amounts of prostacyclin in vitro (medians 0.11 and 0.19 ng/mg tissue respectively), but the segments from the diabetics with nil or minimal retinopathy produced less than those from the diabetics with proliferative retinopathy (medians 0.09 and 0.18 ng/mg respectively). Preoperative plasma immunoreactive concentrations of 6-keto-prostaglandin F1 alpha were not significantly different between the controls and the diabetics (medians 101 and 116 pg/ml respectively). In a separate study, however, 11 diabetics with duration of disease of over 10 years and nil or minimal retinopathy had significantly lower concentrations than a matched group of 16 with background or proliferative retinopathy (medians 79 and 121 pg/ml respectively). These results do not support an association between reduced prostacyclin production and diabetic retinopathy.     

Stimulation of embryonic mouse limb bud mesenchymal cell growth by peptide growth factors

The possible role of peptide growth factors in mammalian intrauterine cell growth has been investigated using primary cultures of undifferentiated mesenchymal cells from 11-day mouse embryo limb buds. When grown as monolayer cultures, proliferation is greatly favored by high cell densities. In medium containing 0.2% serum, purified epidermal growth factor (EGF), fibroblast growth factor (FGF), multiplication stimulating activity (MSA), insulin, and somatomedin-C (Sm-C) do not increase cell growth, but a 30-40,000 molecular weight component of mouse fetal liver conditioned medium is stimulatory. On the other hand, when limb bud cells are grown as high density or micromass cultures, a method which better approximates in vivo growth conditions, all of the purified growth factors tested stimulate cell growth significantly. These growth factors have additive effects when used in combination, the best stimulation being observed with liver medium (10% v/v), EGF (10 ng/ml), FGF (200 ng/ml), and either insulin (1 microgram/ml) or Sm-C (20 ng/ml). We conclude that the response of limb bud cells to growth stimulation is influenced by the manner in which the cells are cultured and that at least four different growth factors are required for optimal in vitro proliferation. One of these, the active component of liver medium, appears to be a previously uncharacterized growth factor.     

Androgen receptor expression in primary prostate cancers of lobund-wistar rats and in tumor-derived cell lines

Summary Prostate tumors were induced in Lobund-Wistar rats by treatment with N-methyl-N-nitrosourea (MNU) and testosterone propionate (TP). Androgen receptor (AR) expression was confirmed in 16 (100%) of the primary prostate cancers, with strong uniform staining in well-differentiated tumors and more variable AR immunoreactivity in poorly differentiated tumors. Epithelial cell lines were established from nine of the tumors. At early passages, four of the tumor cell lines tested were strongly immunoreactive for AR; however, only two of the cell lines, E2(A) and F2, have remained AR-positive. These cell lines specifically bind ³H-DHT at 40 and 19 fmol/mg protein, respectively, and express a 110 kDa AR immunoreactive protein. Proliferation in in vitro culture of both E2(A) and F2 cells was increased in the presence of 5α-dihydrotestosterone (DHT). The antiandrogen, hydroxyflutamide was able to prevent the DHT-induced growth of E2(A) but not F2 cells. Furthermore, hydroxyflutamide alone increased proliferation of F2 cells, suggesting that the androgen signalling pathway in this cell line may be abnormal. Tumorigenicity of the AR-expressing and nonexpressing cell lines was confirmed by xenograft formation following subcutaneous inoculation into intact male nude mice. In summary, carcinogen-induced prostate tumors of Lobund-Wistar rats express AR and two of nine cell lines derived from the tumors express AR. Further evaluation of AR structure in primary prostate tumors forming spontaneously or following MNU and TP induction will determine whether, as in human prostate cancers, disease progression in Lobund-Wistar rats is associated with mutations in the AR gene.     

Dynamic regulation of uncoupling protein 2 content in INS-1E insulinoma cells

Uncoupling protein 2 (UCP2) regulates glucose-stimulated insulin secretion in pancreatic beta-cells. UCP2 content, measured by calibrated immunoblot in INS-1E insulinoma cells (a pancreatic beta-cell model) grown in RPMI medium, and INS-1E mitochondria, was 2.0 ng/million cells (7.9 ng/mg mitochondrial protein). UCP2 content was lower in cells incubated without glutamine and higher in cells incubated with 20 mM glucose, and varied from 1.0-4.4 ng/million cells (2.7-14.5 ng/mg mitochondrial protein). This dynamic response to nutrients was achieved by varied expression rates against a background of a very short UCP2 protein half-life of about 1 h.     

Hormonal studies in a patient with PP-producing tumors

Five years after the removal of pure pancreatic polypeptide (PP) producing tumors, concentrations of circulating levels of PP, insulin, glucagon, and growth hormone in the basal state, after insulin-induced hypoglycemia, and after a protein-rich meal were determined in a patient with previous truncal vagotomy and Billroth II gastrectomy. Basal plasma levels of PP ranged between 2180 and 2660 pg/ml suggesting persistence or recurrence of PP producing tumors. Concentrations of the other hormones were within normal values. After insulin injection (0.1 U/Kg) levels of PP and glucagon were not modified while those of GH rose from 3.2 to 22.6 ng/ml. After a protein meal (450 gms. of cooked ground beef meat) a sharp rise of plasma PP was observed to a peak of 11310 pg/ml at 10 min. Moreover, plasma levels of immunoreactive insulin also showed an equally prompt rise to a peak of 532 microU/ml while plasma glucagon rose simultaneously to 448 pg/ml. The cause of the abnormal PP, insulin and glucagon responses could not be ascertained but we postulate that they are derived from pancreatic tumors of mixed cell type.     

Identification of Insulin in Chick Embryo Retina During Development and Its Inhibitory Effect on DNA Synthesis

Incubation of chick embryo retinal explants with insulin resulted in a pronounced inhibition of thymidine uptake and incorporation into trichloroacetic acid-insoluble fraction. The inhibitory effect was highest with explants from embryos at day 7 and day 8, and thereafter it declined markedly with the age of embryos until day 11. A time-course study of the effect revealed that the inhibition occurred after a lag time; both thymidine uptake and incorporation were not altered significantly after 2-6 h of incubation with insulin, but began to decrease thereafter, reaching the maximum after 16 h. The effect was also dose dependent. After 16 h of incubation, the maximal inhibition (65%) was found with 10(-8) M insulin. Insulin caused similar effects also on thymidine kinase activity. All these effects were obtained by using minimal essential medium without glutamine. The addition of glutamine to the medium reduced the inhibitory effect of insulin. Retinas of chick embryos contain immunoreactive insulin. Retinal immunoreactive insulin was at the highest level (1.12 ng/mg of protein) in the youngest retinas studied (day 6), then it declined with age, reaching the lowest value (0.58 ng/mg of protein) at day 14. This value did not vary significantly during the third week of development. A potential biological role of insulin in retinal development is discussed.     

Cytosolic levels of an estrogen-induced breast cancer-associated peptide (TFF1/pS2) in colorectal cancer: clinical significance and relationship with steroid receptors

BACKGROUND: The Trefoil Factor 1 (TFF1/pS2), a peptide consisting of 60 amino acids, is the most abundant estrogen-induced messenger RNA in MCF-7 breast cancer cells and is also expressed by colorectal carcinomas. The objective of this work was to evaluate the cytosolic TFF1 content in colorectal carcinomas, its possible relationship with estrogen and progesterone receptors as well as with clinicopathological tumor parameters, and its potential prognostic significance. METHODS: Cytosolic TFF1 levels were examined by immunoradiometric assay in 178 patients with resectable colorectal cancer. The mean follow-up period was 32 months. RESULTS: There was a wide variability of cytosolic TFF1 levels in tumor-surrounding mucosa samples (0.09-42.5 ng/mg protein) as well as in tumors (0.01-270 ng/mg protein). Comparison of paired mucosa and carcinoma samples showed significantly higher TFF1 levels in tumors (mean: 17.1 ng/mg protein) than in mucosa samples (10 ng/mg protein) (p = 0.027). TFF1 levels were significantly higher in mucosa samples surrounding distal colon and rectal tumors (p = 0.0001) and in tumor samples obtained from older patients (p = 0.007). However, there were no significant differences in tumor TFF1 levels with respect to clinicopathological parameters such as the patient's sex, tumor location, stage, histological grade, ploidy, S-phase, or tumor estrogen and progesterone receptors. In addition, there was no significant relationship between tumor TFF1 levels and disease outcome. CONCLUSIONS:TFF1 may play an as yet undetermined role in the tumorigenesis of colorectal carcinomas. However, cytosolic levels of TFF1 do not seem to have any prognostic significance in colorectal carcinomas.     

Processing of mutated human proinsulin to mature insulin in the non-endocrine cell line,CHO

Heterologous genes encoding proproteins, including proinsulin, generally produce mature protein when expressed in endocrine cells while unprocessed or partially processed protein is produced in non-endocrine cells. Proproteins, which are normally processed in the regulated pathway restricted to endocrine cells, do not always contain the recognition sequence for cleavage by furin, the endoprotease specific to the constitutive pathway, the principal protein processing pathway in non-endocrine cells. Human proinsulin consists of B-Chain — C-peptide — A-Chain and cleavage at the B/C and C/A junctions is required for processing. The B/C, but not the C/A junction, is recognised and cleaved in the constitutive pathway. We expressed a human proinsulin and a mutated proinsulin gene with an engineered furin recognition sequence at the C/A junction and compared the processing efficiency of the mutant and native proinsulin in Chinese Hamster Ovary cells. The processing efficiency of the mutant proinsulin was 56% relative to 0.7% for native proinsulin. However, despite similar levels of mRNA being expressed in both cell lines, the absolute levels of immunoreactive insulin, normalized against mRNA levels, were 18-fold lower in the mutant proinsulin-expressing cells. As a result, there was only a marginal increase in absolute levels of insulin produced by these cells. This unexpected finding may result from preferential degradation of insulin in non-endocrine cells which lack the protection offered by the secretory granules found in endocrine cells.     

Further characterization of a CD99-related 21-kDa transmembrane protein (VAP21) expressed in Syrian hamster cells and its possible involvement in vesicular stomatitis virus production

The VAP21, a CD99-related 21-kDa transmembrane protein, was first detected in the enveloped virions that were grown in a Syrian hamster-derived cell line, BHK-21 (Sagara et al., 1997; Yamamoto et al., 1999). We further tried to elucidate the nature and properties of VAP21. The VAP21 was detected in various organs of the Syrian hamster as well as in the Syrian hamster-derived cell lines (BHK-21 and HmLu-1). We could not detect the VAP21 antigen in other cell lines derived from other animal species we examined, including a Chinese hamster (CHO-K1), mouse (neuroblastoma C1300, clone NA), dog (MDCK), monkey (COS-7), and human (HeLa, HepG2). We tried to introduce the VAP21 gene into VAP21-negative cell lines using a tetracycline-regulated gene expression system. All of our trials, however, resulted in failure to establish stably positive inducible cell lines. To the contrary, we could easily establish the VAP21-overexpressing cell lines from the Syrian hamster cell lines, which were successfully grown and maintained without any loss of VAP21 expression even under the induced culture conditions. In such VAP21-overexpressing cells, production of the vesicular stomatitis virus (VSV) was increased several-fold, while suppression of the VAP21 expression resulted in reducing the VSV yields. From these results, we conclude that the VAP21 is a physiologically active cell membrane component of some animal species including the Syrian hamster, and might positively be involved in the VSV replication.     

Failure of IGF-1 to affect protein turnover in muscle from growth-retarded neonatal rats

To investigate the response of the growth retarded neonatal rat to insulin-like growth factor-I (IGF-I) we have measured the effect of IGF-I on in vitro muscle protein synthesis and degradation rates in growth retarded and control neonatal rat pups. The growth retarded pups were growth retarded in utero by ligation of the uterine blood supply at day 17 of gestation. Basal levels of muscle protein synthesis in vitro were significantly lower in growth retarded pups compared with controls. Protein degradation rate were not different in muscles taken from the two groups. IGF-I stimulated protein synthesis in muscle from control pups by 12% and 15% at 20 ng/ml and 200ng/ml respectively. Net protein degradation was inhibited by 20% in the presence of 20ng/ml IGF-I. IGF-I had no effect on net protein synthesis or degradation in muscle from growth retarded pups. Neither Multiplication Stimulating Activity (at 20ng/ml or 200ng/ml) nor insulin (at 40ng/ml or 800ng/ml) was able to increase synthesis or decrease degradation of protein. Specific receptors for IGF-I are present on muscle membranes from both groups. Unlabelled IGF-I was more effective than MSA or insulin in competing with 125I-IGF-I for binding to the receptor. The relative affinities are consistent with type I IGF receptors. The affinity of these receptors for IGF-I was similar (Kd approximately 5nM) in both groups and the receptor concentration in both cases was approximately 250 fmol/mg protein. The refractility of tissue from growth retarded pups to IGF-I may be partially responsible for the lack of catch up growth in growth retarded neonates.     

Developmental and ultrastructual characteristics of mouse oocytes grown <Emphasis Type="Italic">in Vitro</Emphasis> from primordial germ cells

The aim of this study was to clarify the developmental and ultrastructual characteristics of oocytes grown in vitro from primordial germ cells. The female genital ridges at 12.5 days post coitus were cultured for 18 days on an insert membrane in Waymouth's MB752/1 medium, supplemented with 15% fetal bovine serum and 1 mM sodium pyruvate; subsequently, the follicles isolated from the tissue were cultured for eight days in Waymouth's medium supplemented with 5 microg/ml insulin, 5 microg/ml transferrin, 5 ng/ml selenium, 10 mIU/ml follicle stimulating hormone, and 100 ng/ml stem cell factor. The primordial germ cells developed in vitro into oocytes of more than 60 microm in diameter. The transmission electron microscopic analysis indicated that the oocytes, which developed in vitro, showed no obvious abnormality in their ultrastructure and had organelles appropriate for the oocyte size. However, a delay in the progressive changes of morphology in some of the organelles during oocyte growth was often found when comparing them to oocytes grown in vivo.     

Induction of cholesteryl ester transfer protein in adipose tissue and plasma of the fructose-fed hamster

Cholesteryl ester transfer protein (CETP) plays a pivotal role in the reverse transport of cholesterol and in the remodeling of circulating lipoproteins. While plasma and adipose tissue levels of CETP are affected by a variety of metabolic conditions, the extent of the effects of dietary factors, other than high cholesterol feeding, are not well understood. To further explore this paradigm, male Golden Syrian hamsters were fed for 4 weeks with a 60%-enriched fructose diet (F) and were compared to a matched group of animals fed with a normal chow diet (N). After feeding for 4 weeks, plasma insulin concentrations were lower in animals fed fructose than in control animals (F: 3.3+/-0.8 vs N: 7.4+/-1.9 ng/mL; p<0.03), but there was no significant difference in plasma glucose concentrations between the two groups (F: 138+/-7 vs N: 148+/-10 mg/dL; p>0.05). Fructose-fed animals showed significant increases in plasma triglyceride (F: 269+/-22 vs N: 165+/-22 mg/dL; p<0.01) and plasma cholesterol (F: 150+/-10 vs N: 113+/-6 mg/dL; p<0.02) concentrations compared with control animals. Total CETP activity and immunoreactive mass were higher in the plasma of fructose-fed animals that in that of controls (F: 1036+/-70 vs N: 826+/-43 pmol/h/mL, p<0.04 and F: 24.5+/-3.1 vs N: 37.5+/-4.3 AU, p<0.02, respectively). Adipose tissue CETP mRNA levels, assessed by the very sensitive ribonuclease protection assay, were 53% higher in fructose-fed animals than in controls (F: 14.1+/-2.0 vs N: 9.2+/-1.0 AU over a rRNA control; p<0.04). Adipose tissue CETP activity and immunoreactive mass also showed a statistically significant increase in the fructose-fed hamsters compared with those fed a normal diet (p<0.04). In conclusion, fructose feeding in Syrian hamsters induces a mixed dyslipidemia. These metabolic changes are accompanied by a significant increase in CETP levels, both in plasma and in adipose tissue. This phenomenon suggests that the increase in the expression of adipose tissue CETP may be caused either by the ambient hypercholesterolemia resulting from fructose feeding or by an attenuation of a possible inhibitory effect of plasma insulin concentrations on the expression of adipose tissue CETP in this feeding paradigm.     

Effect of cholecystokinin, secretin, and gastric inhibitory polypeptide on insulin release from the isolated perfused rat pancreas

The actions of gastric inhibitory polypeptide (GIP) on insulin release from the isolated perfused rat pancreas were compared with those of pure secretin and cholecystokinin (CCK). At dose levels physiologically achievable for GIP (1 ng/mL perfusate), infusions of CCK stimulated significant insulin release both on a weight (1 ng/mL) and a molar (770 pg/mL) basis. Although 50% as potent as GIP on a weight basis and 43% as potent on a molar basis, the insulin response to CCK was multiphasic and sustained for the duration of the infusion. The action of CCK, like that of GIP, was glucose dependent yielding no significant insulin release at a low perfusate glucose concentration (80 mg/dL). Irrespective of perfusate glucose concentration or dose (1 or 5 ng/mL), secretin failed to stimulate significant release of insulin from the perfused pancreas. It was concluded that secretin is ineffective as an incretin and that a physiological role for CCK in an enteroinsular axis awaits accurate measurement of circulating levels of immunoreactive CCK.     

Tumorspheres but Not Adherent Cells Derived from Retinoblastoma Tumors Are of Malignant Origin

Verification that cell lines used for cancer research are derived from malignant cells in primary tumors is imperative to avoid invalidation of study results. Retinoblastoma is a childhood ocular tumor that develops from loss of functional retinoblastoma protein (pRb) as a result of genetic or epigenetic changes that affect both alleles of the RB1 gene. These patients contain unique identifiable genetic signatures specifically present in malignant cells. Primary cultures derived from retinoblastoma tumors can be established as non-adherent tumorspheres when grown in defined media or as attached monolayers when grown in serum-containing media. While the RB1 genotypes of tumorspheres match those of the primary tumor, adherent cultures have the germline RB1 genotype. Tumorspheres derived from pRb-negative tumors do not express pRb and express the neuroendocrine tumor markers synaptophysin and microtubule-associated protein 2 (MAP2). Adherent cells are synaptophysin-negative and express pRb, the epithelial cell marker cytokeratin that is expressed in the retinal pigmented epithelium and the vascular endothelial cell marker CD34. While tumorspheres are of malignant origin, our results cast doubt on the assumption that adherent tumor-derived cultures are always valid in vitro models of malignant cells and emphasize the need for validation of primary tumor cultures.     

Insulin activates the Raf-1 protein kinase

Several growth factors and mitogens have been shown to activate the proto-oncogene product Raf-1 protein kinase in murine fibroblasts, apparently through a direct agonist-stimulated tyrosine phosphorylation of the Raf-1 protein. We investigated the possibility that insulin could also activate the Raf-1 kinase, since its receptor also contains an intrinsic insulin-activated protein tyrosine kinase activity. In several cell lines expressing relatively large numbers of insulin receptors, insulin rapidly stimulated the phosphorylation of immunoreactive Raf-1 protein. In H35 cells, a line of well differentiated rat hepatoma cells, the effect of insulin was maximal by 6 min and at 7 nM insulin and occurred normally in cells virtually completely depleted of protein kinase C activity. The insulin-stimulated increase in Raf-1 protein phosphorylation occurred concurrently with a 3-fold increase in Raf-1 protein kinase activity. However, phosphoamino acid analysis showed that only phosphoserine and a trace of phosphothreonine were present in the Raf-1 protein after insulin stimulation of the cells. This was true even when investigated at shorter times (4 min) after insulin stimulation and despite the use of phosphotyrosine phosphatase inhibitors. We conclude that insulin can rapidly activate the Raf-1 kinase in some insulin-sensitive cell types but that this activation probably occurs through a mechanism distinct from direct phosphorylation of the Raf-1 protein by the insulin receptor protein tyrosine kinase.     

Determination of immunoreactive endothelin in medium from cultured endothelial cells and human plasma

We have developed a sensitive and selective radioimmunoassay for porcine/human endothelin (ET1). The assay has a detection limit of 0.62 pg/tube and exhibits no cross-reactivity to atrial natriuretic peptide, arginine vasopressin, or angiotensin II. Procedures were developed for extraction of endothelin from human plasma samples and samples of buffer from endothelial cell incubations using C18 Sep-Pak extraction cartridges. The mean recovery following extraction was approximately 80%. Both bovine and porcine aortic endothelial cells were found to produce immunoreactive endothelin (IR-ET) with porcine cells producing 4.7 +/- 1.1 ng of IR-ET/mg cell protein after 6 hours. Human plasma samples were extracted, assayed and found to contain a mean concentration of 2.0 +/- 0.4 pg/ml of IR-ET.     

Evidence for the presence of immunoreactive histidyl-proline diketopiperazine [Cyclo (His-Pro)] in the adult human brain

The current study was undertaken to evaluate the presence of cyclo (His-Pro) in adult human brain tissues obtained at autopsy. We found evidence for immunoreactive cyclo (His-Pro), which diluted in parallel to the radioimmunoassay standard curve and which had mobility on HPLC that was similar to synthetic cyclo (His-Pro), in several regions of the adult human brain. Whereas the levels of cyclo (His-Pro) in the pituitary stalk-median eminence were high (2.2 ng/mg protein), the concentrations in the whole hypothalamus were much lower (0.105 ng/mg protein). Among the extrahypothalamic brain regions examined, the levels of cyclo (His-Pro) were highest in the cerebellar hemisphere (0.168 ng/mg protein) and olfactory bulbs (0.180 ng/mg protein) and were lowest in the hippocampus (0.080 ng/mg protein) and occipital cortex (0.079 ng/mg protein). Thus, immunoreactive cyclo (His-Pro) has widespread distribution in the adult human brain and the potential exists for this cyclic diepeptide to play a role in human brain function.