The distribution of DNA among dividing mitochondria of Tetrahymena pyriformis.

A squash technique was developed for log phase Tetrahymena pyriformis which permitted the resolution of over 100 individual mitochondria from a single cell. Mitochondria incorporated thymidine at all stages of the cell cycle, even when nuclear DNA synthesis was not occurring. During the stage of macronuclear DNA synthesis, however, there was a significant increase in the extent of mitochondrial labeling. Low radioautograph background suggests that mitochondrial DNA is synthesized at the mitochondria themselves. All mitochondria incorporated thymidine-3H within one population-doubling time. Grain counts also showed that the amount of mitochondrial label was retained for four generations and that this label remained randomly distributed among all mitochondria during this time. The results are not consistent with any theory of de-novo or "microbody" origin of mitochondria, but do support the hypothesis that mitochondria are produced by the growth and division of preexisting mitochondria. The stability of the mitochondrial DNA and its distribution among daughter mitochondria satisfy two prerequisites for a genetic material. The possibility is discussed that some of the genetic information for the mitochondrion is contained in the DNA associated with this organelle.

Effects of 17 beta-estradiol and medroxyprogesterone acetate upon MtTW15 mammosomatotropic pituitary tumor growth and hormone production in male and female rats.

The purpose of this study was to characterize the effects of two functionally diverse steroids, 17 beta-estradiol and medroxyprogesterone acetate (MPA), on MtTW15 rat mammosomatotropic pituitary tumor growth and hormone production. Steroid responsiveness, as well as the hormonally autonomous nature of the tumor, was studied by treating both male and female tumor-bearing rats for 7 weeks with weekly injections of either 17 beta-estradiol (600 ng/g body weight/week) or MPA (200 microgram/g body weight/week) and, subsequently, comparing both the tumor weights and the in vivo production of growth hormone (GH) and prolactin (PRL) among the treatment groups. Large tumors (6 to 20 gm) were obtained in all treatment groups, indicating hormonal autonomy; however, tumors were markedly smaller, on the average, in untreated males an ovariectomized females. Treatment of such rats with 17 beta-estradiol stimulated tumor growth. Radioimmunoassay of tumor and serum GH and PRL levels in all treatment groups indicated the following: (a) tumors from untreated male or female hosts did not favor the production of one hormone over the other to any great extent; (b) MPA, however, promoted significant increases (p less than 0.05) in GH production in both male and female tumor-bearing rats while having little effect on the production of PRL; and (c) 17 beta-estradiol significantly inhibited (p less than 0.05) GH production and promoted PRL production by tumors borne by either sex. Selected studies utilizing multiple doses of MPA (1 to 500 microgram per gm body weight per week) and 17 beta-estradiol (10 to 800 ng per gm body weight per week) were accomplished and demonstrated that hormone production can be influenced in a dose-related manner. These results indicated that the estrogen-induced MtTW15 rat pituitary tumor is hormonally autonomous, yet divergently responsive to two different classes of steroidal compounds, thus making this tumor line an appropriate model for the study of hormonally responsive pituitary tumor cells.

PIP: The purpose of this study was to characterize the effects of 2 functionally diverse steriods, 17beta-estradiol and (MPA) medroxyprogesterone acetate on MtTW15 rat mammosomatotropic pituitary tumor growth and hormone production. Steroid responsiveness, as well as the hormonally autonomous nature of the tumor, was studied by treating both male and female tumor-bearing rats for 7 weeks with weekly injections of either 17beta-estradiol (600 ng/g body weight/week) or MPA (200 mcg/g body weight/week) and, subsequently, comparing both the tumor weights and the in vivo production of (GH) growth hormone and (PRL) prolactin among the treatment groups. Large tumors (6 to 20 gm) were obtained in all treatment groups, indicating hormonal autonomy; however, tumors were markedly smaller, on the average, in untreated males and ovariectomized females. Treatment of such rats with 17beta-estradiol stimulated tumor growth. Radioimmunoassay of tumor and serum GH and PRL levels in all treatment groups indicated the following: (a) tumors from untreated male or female hosts did not favor the production of 1 hormone over the other to any great extent; (b) MPA, however, promoted significant increases (p 0.05) in GH production in both male and female tumor-bearing rats while having little effect on the production of PRL; and (c) 17-estradiol significantly inhibited (p 0.05) GH production and promoted PRL production by tumors borne by either sex. Selected studies utilizing multiple doses of MPA (1 to 500 mcg/gm body weight/week) and 17 beta-estradiol (10 to 800 ng/gm body weight/week) were accomplished and demonstrated that hormone production can be influenced in a dose-related manner. There results indicated that the estrogen-induced MtTW15 rat pituitary tumor is hormonally autonomous, yet divergently responsive to 2 different classes of steroidal compounds, thus making this tumor line an appropriate model for the study of hormonally responsive pituitary tumor cells.

Carcinogenic activities of various steroidal and nonsteroidal estrogens in the hamster kidney: relation to hormonal activity and cell proliferation.

The therapeutic use of estrogens has been associated with an increased risk of some of the most predominant, as well as less prevalent, cancers in women. The estrogen-induced renal tumor is one of the primary animal models to evaluate the carcinogenic properties of estrogens. Correlations were made with various estrogens by using parameters of estrogenicity end points such as competitive binding, progesterone receptor induction, and alterations in prolactin levels; in vitro renal proximal cell proliferation; and in vivo estrogen-induced carcinogenicity. The most potent estrogens were Moxestrol (MOX), diethylstilbestrol (DES), and 17 beta-estradiol, followed by indenestrol B, 16 alpha-hydroxyestrone, and 11 beta-methoxyestradiol with moderate estrogenic activities, whereas 11 beta-methylestradiol, 17 alpha-estradiol, indanestrol, and deoxoestrone were all relatively weaker. As expected, hydrolyzed Premarin (unconjugated estrogens) was strongly estrogenic. Of the estrogens tested, MOX was the most potent carcinogenic estrogen in the hamster kidney. Both 16 alpha-hydroxyestrone and 11 beta-methoxyestradiol induced intermediate tumor incidences with distinctly lower frequencies of renal tumor foci compared to the most potent carcinogenic estrogens. However, hamsters treated for 9.0 months with 11 beta-methylestradiol, 17 alpha-estradiol, deoxoestrone, and indanestrol exhibited no tumors. In contrast, treatment with estrone, equilin plus d-equilenin, and hydrolyzed Premarin for the same time period resulted in 100% renal tumor incidences and numerous tumor foci. Cell proliferation studies of cultured hamster kidney proximal tubule cells were carried out at varying estrogen concentrations (0.01-100 nM). Exposure to MOX resulted in consistently high renal cell proliferative response over a concentration range of 0.1-10 nM. Strongly carcinogenic estrogens such as estrone had a maximal renal cell proliferation response (2.4-fold above untreated control levels) between 0.1 and 10 nM, DES and 17 beta-estradiol responded at 1.0 nM, and 4-hydroxyestradiol responded at 10 nM. Interestingly, exposure to ethinylestradiol, a potent estrogen, at similar or higher doses as those used for DES and 17 beta-estradiol, yielded only a 10% renal tumor incidence and induced only a 1.7-fold increase in proximal tubule cell proliferation. In contrast, 17 alpha-estradiol, deoxoestrone, indanestrol, and 11 beta-methylestradiol, all weakly estrogenic and noncarcinogenic agents, had relatively little effect on tubule cell proliferation. The hydrolyzed Premarin exhibited a maximal 2.0-fold cell proliferative response at 10 nM. The present results provide clear evidence that, in the hamster kidney, the degree of carcinogenicity of a given estrogen correlates with its ability to induce proximal tubule cell proliferation in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Relative carcinogenic activity of various synthetic and natural estrogens in the Syrian hamster kidney.

Both synthetic and natural estrogens have been studied for their ability to induce renal carcinomas in castrated male hamsters after 9.0 months of treatment. Tumor foci were detected in frozen serial sections stained histochemically for estrase activity. Both diethylstilbestrol (DES) and 17 beta-estradiol had equal ability (100%) to induce renal tumors [approximately 20.5 +/- 3 (S.E.) tumor foci] in these animals. Hexestrol induced the same incidence and number of renal carcinoma foci as DES or 17 beta-estradiol. However, alpha -dienestrol and DES 3,4-oxide showed an 86 to 88% incidence of renal tumors in hamsters (approximately 10.8 +/- 3). When equilin and d-equilenin, components of therapeutic conjugated estrogens, were tested, only equilin had a 76% incidence of renal tumor foci (5.5 +/- 0.9). The ability of these stilbene and steroidal estrogens to compete for renal tumor estrogen receptor generally correlated well with their ability to cause renal tumorigenesis in the hamster with one notable exception. Although ethinyl estradiol competed as well as did DES or 17 beta-estradiol for estrogen receptor, had similar ability to induce renal progesterone receptor, and led to similar high serum prolactin levels as either DES or 17 beta-estradiol, it had only weak carcinogenic activity (21%) in the hamster kidney (0.6 +/- 0.5 foci). These data represent the first detailed analysis of the relative carcinogenic activity of different estrogens within a given tumor-inducing system, and based on the carcinogenicity data of hexestrol and alpha-dienestrol presented herein, they suggest that epoxidation of the olefinic double bond and the p-quinone metabolite of DES probably are not involved significantly in its carcinogenic activity. Moreover, the poor carcinogenic activity of ethinyl estradiol in this system, despite strong estrogenicity, suggests that estronic activity alone may not be sufficient to effect renal tumorigenesis in the hamster.

Insulin secretion in mammosomatotropic tumor-bearing and pregnant rats. A role for lactogens.

The experiments reported here investigate the effects of two conditions of elevated lactogen activity on the threshold of glucose stimulation of insulin secretion and suprathreshold, glucose-stimulated insulin secretion in the isolated, perfused rat pancreas. In both the tumor-bearing animals and pregnant animals, the glucose threshold for insulin release was markedly reduced and the suprathreshold insulin release was elevated over that observed in control pancreata. There was no change on the glucose threshold or extent of release of somatostatin secretion. Altered insulin secretion appears to be readily reversible, since the secretion profile from postlactating animals was not different from the controls. The possible mechanistic role of lactogens is discussed.

Effect of MtTW15 mammosomatotropic tumors on pancreatic islet hormones.

The effects of hypersecretion of growth hormone and prolactin on islet endocrine cells have been studied by radioimmunoassays, immunocytochemistry, and morphometry in randomized samples of pancreata from MtTW15 mammosomatotropic tumor-bearing and control rats. The randomized sampling procedure, validated by immunoassays, allowed evaluation of both hormone content (immunoassay) and endocrine cell population (immunocytochemistry) on samples derived from the same origin. Hyperinsulinemia (2x) and non-fasting hypoglycemia in 10-wk-tumor rats were normalized 3 wk after tumor removal. Pancreatic weight was doubled, but proportional to body weight increases. Islet/pancreas ratio was constant (1.29 +/- 0.05%) and the same in tumor, tumor-removed, and control animals, but average islet dimensions were increased by 30% and average area doubled in tumor animals. Frequency analysis showed fewer small (less than 70 micrometers) and more large (greater than 140 micrometers) islets in tumor animals, but no change in average islet shape shown by average axis ratios of 1.4 in all groups. Pancreatic content of insulin and glucagon was doubled, while that of somatostatin was constant. These changes were not completely reversed in tumor-removed animals. Similarly, a significant doubling in islet-derived mass was mainly due to a doubling of the B-cell mass as the average proportion of endocrine cells per islet shifted from 66%, 26%, and 18% to 81%, 18%, and 3% for B-, A-, and D-cells of control and tumor-bearing rats, respectively. Immunocytochemically detectable insulin was found in duct cells of tumor animals, but not controls. Whether such cells represent a functional reserve remains to be determined.

Increased dye coupling in pancreatic islets from rats in late-term pregnancy.

Our previous studies have suggested that elevated lactogen, increased glucose-stimulated insulin secretion, and increased beta-cell coupling are associated. To determine whether this association occurs under conditions of physiologically increased lactogen, we have studied the extent of dye coupling in rat islets during the later stage of pregnancy. These animals have high plasma lactogen levels in the form of placental lactogen, increased plasma insulin, and decreased plasma glucose. The fluorescent tracer, Lucifer yellow CH, was microinjected into central cells of islets from both pregnant and virgin rats, and the extent of transfer was quantitated by determining the projected area of dye spread. Two area measurements were made for each injection, one around the entire discernible fluorescent region ("outer") and another around the distinct brighter region of cells surrounding the injected cell ("inner"). Pregnancy increased dye transfer, as determined by both measurements. The outer area of dye transfer was 9047 +/- 775 microns2 for the islets from pregnant rats and 4699 +/- 391 microns2 for the islets from virgin rats (P less than .001). Similarly, pregnancy increased the inner area of dye transfer, 1447 +/- 161 microns2 for the islets from pregnant rats and 795 +/- 80 microns2 for the islets from virgin rats (P less than .001). These results support the hypothesis that elevated lactogen, increased glucose-stimulated insulin secretion, and increased beta-cell dye coupling are associated under physiological conditions. The study indicates that enhanced beta-cell coupling is part of the structural and functional adaptation that the islets undergo during a subject's pregnancy and demonstrates that the extent of beta-cell coupling is regulated by a physiological condition.

Prolactin enhances cell-to-cell communication among beta-cells in pancreatic islets.

To determine the role of prolactin in increasing junctional communication among islet beta-cells, we studied dye coupling in pancreatic islets exposed to elevated levels of prolactin in vivo and in vitro. Islets were isolated from rats immediately after lactation or from rats bearing mammosomatotropic tumors (MtTW15), conditions involving high levels of prolactin (either 5-fold or 1000-fold control levels, respectively). When beta-cells were microinjected with the gap junction permeant dye Lucifer yellow CH, the mean number of dye-coupled cells per injection was approximately 10-fold greater than in islets from virgin control rats. As a more direct test of the effects of prolactin on beta-cell coupling, islets isolated from virgin rats were treated for 90 min with 500 ng/ml rat prolactin in the presence of low glucose (2.8 mM) and were microinjected with dye. The mean number of dye-coupled cells per injection increased by 6.7-fold over controls with low glucose, demonstrating a direct effect of prolactin on beta-cell coupling. In vitro treatment with high glucose (16.7 mM) resulted in a 2.7-fold increase in dye-coupled cells per injection. We discuss the possible relationship between the effects of glucose and of prolactin on coupling.

Prolonged hypoglycemic effect in diabetic dogs due to subcutaneous administration of insulin in liposomes.

The biologic action of insulin entrapped in liposomes (phospholipid vesicles) has been investigated following subcutaneous injection to dogs made diabetic with a combination of alloxan and streptozotocin. The fats of the liposomally entrapped material was determined by injecting rats subcutaneously with either 125I-insulin or the labeled polysaccharide 14C-inulin, incorporated in liposomes labeled with 3H-cholesterol. Injection of liposome insulin (0.75 U/kg) to five diabetic dogs resulted in a mean (+/- SEM) blood glucose fall from 16.4 +/- 0.8 to 2.9 +/- 0.4 mmol/L. The glucose level had still not returned to baseline after 24 h and, correspondingly, immunoreactive insulin (IRI) could still be detected in frozen and thawed plasma 24 h after injection. In contrast, the hypoglycemic effect of the same dose of free insulin with or without empty liposomes virtually ended within 8 h and IRI levels returned to baseline by 3 h after injection. In experiments on rats with liposomally entrapped 125I-insulin or 14C-inulin the proportion of the injected dose of tracer recoverable by excision of the injection site remained constant after about 1 h and 70% of the dose was still fixed in subcutaneous tissue for at least 5 h thereafter. When the plasma collected 3 h after subcutaneous injection of labeled liposomes containing 125I-insulin was passed through a column of Sepharose 6B, 50-75% of the 125I-activity was found in the fractions associated with intact liposomes. One possibility for the persistence of the hypoglycemic effect and of measurable IRI following injection of liposome insulin could be the presence of intact liposomes in the circulation for many hours after adsorption had ceased.

Regulation of islet beta-cell proliferation by prolactin in rat islets.

This study examined the effects of prolactin on beta-cell proliferation in pancreatic islet of Langerhans. Insulin secretion and beta-cell proliferation were significantly increased from neonatal rat islets cultured for 4 days in the presence of either 500 ng/ml ovine prolactin (oPRL) or rat prolactin (rPRL). These effects could be prevented by including anti-oPRL serum in the culture media. Although insulin secretion and beta-cell proliferation were slightly higher during the first 24 h of exposure to rPRL, maximal response was observed after 4 days for insulin secretion and 6-10 days for beta-cell proliferation. The initial mitogenic response of beta-cell to rPRL occurred by the limited recruitment of nondividing beta-cells into the cell cycle and by most daughter cells proceeding directly into additional cell division cycles. Subsequently, the maximal effect of rPRL on beta-cell proliferation was maintained by a higher rate of recruitment of previously nondividing beta-cells into cell cycle with only one fourth of the daughter cells continuing to divide. These observations are difficult to reconcile with the proposal that a limited pool of beta-cells capable of undergoing cell division exists in islets. Instead, these observations suggest that individual beta-cells are transiently re-entering the cell cycle and dividing infrequently in response to rPRL. In this case, the majority of the beta-cells would not be expected to be in an irreversible Go phase. We also demonstrated that the effects of rPRL on beta-cell proliferation occur at normal serum glucose concentrations and are affected by inhibitors of polyamine metabolism. Additional studies on the effects of rPRL on beta-cells should provide important information on the regulation of beta-cell proliferation during conditions of increased insulin demand.

Glucocorticoid-like actions of medroxyprogesterone acetate upon MtTW15 rat mammosomatotropic pituitary tumors.

We have shown recently that the MtTW15 experimental rat pituitary tumor responds to medroxyprogesterone acetate (MPA) by increasing GH production. In this study, using a single saturating dose assay and dextran-coated charcoal separation, the data indicate that the MtTW15 tumor contains cytosolic MPA binding sites. The concentration of sites (approximately 4 pmol/g tumor) was similar for tumors derived from male or female hosts but was significantly reduced in tumors from MPA- or estradiol-treated rats. The MPA binder sedimented to 4S on low salt sucrose density gradients and was of high affinity (Kd = 5.0 +/- 0.4 X 10(-9) M). However, binding specificity studies showed glucocorticoids to be better ligands than MPA. MtTw15 tumors were also analyzed for cytosolic progestin ([3H]R5020) and glucocorticoid ([3H]dexamethasone) binding sites. Only low levels of an estradiol-inducible progestin binder were found. In contrast, the concentration of glucocorticoid binding sites was similar to that observed for MPA, approximately 4 pmol/g tumor, as were the characteristics of the binding, i.e. 5.1 +/- 0.2S, Kd = 4.1 +/- 0.5 X 10(-9) M, and similar binding specificities. Both MPA and estradiol treatment of tumor-bearing rats decreased the concentration of both MPA and glucocorticoid binding sites. Furthermore, studies to determine if glucocorticoids would mimic the in vivo effect of MPA upon MtTW15 tumors, i.e. altered tumor hormone production, supported such a hypothesis. We conclude that the MtTW15 rat pituitary tumor contains a cytosolic glucocorticoid receptor and that MPA can interact with this receptor. The glucocorticoid receptor may be responsible for the MPA- and glucocorticoid-induced alterations in GH production.

Number and size of islets of Langerhans in pregnant, human growth hormone-expressing transgenic, and pituitary dwarf mice: effect of lactogenic hormones.

To determine the effects of lactogenic hormones on pancreatic islet size and numbers, islets of 3-month-old female mice were intravitally stained by an ip injection of an alkaline-alcohol solution of diphenylthiocarbazone (dithizone; 100 micrograms/g BW). After 15 min, animals were killed, and pancreases were removed, diced, cleared in glycerol, and whole mounted on slides. Major and minor axes of Zn dithizoate-stained islets were measured at x40 magnification. Islet areas and volumes were calculated. Animals and appropriate controls studied included 16-day pregnant, two lines of human GH-expressing transgenic, and two lines of pituitary PRL- and GH-deficient dwarf mice. Islet numbers per pancreas ranged from about 500-1200 in all groups except the transgenic mice, in which two of five animals in one group and one of five in the other showed significant increases in islet numbers (> 3 x SD control mean). In all cases, significant (P < 0.05) changes in both islet area and volume occurred. Area increased 2-fold in both pregnant and transgenic mice and decreased by a similar amount in dwarf mice. Islet volume increased 2- and 3-fold in pregnant and transgenic animals, respectively, and decreased 2- to 5-fold in dwarf mice. Analysis of the distributions of islet sizes revealed that almost all of the volume increases in the pregnant and transgenic mice and the decreases in dwarf mice were accounted for by alterations in the numbers and sizes of large (diameter, > 150 microns) islets. Our results with dwarf mice show that maintenance of islet numbers is not dependent upon pituitary PRL or GH; however, results with transgenic mice suggest that prolonged high levels of lactogens may induce islet neogenesis. The islet area and volume results for all of the mice studied support the hypothesis that lactogenic hormones are potent regulators of islet mass.

Effects of cysteamine on pituitary, MTTW15 tumor, and serum prolactin levels measured by rat lymphoma cell bioassay and radioimmunoassay.

Cysteamine (CSH), a sulfhydryl compound, reduces both serum and anterior pituitary (AP) PRL measured by RIA. We have used the Nb2 lymphoma cell bioassay (BIO) for PRL to evaluate possible CSH-related changes in PRL levels in sera and tissues of male and MtTW15 mammosomatotropic tumor-bearing female rats. Experimental animals received a single sc injection of CSH (300 mg/kg), and samples were collected 0.5-24 h later. Since CSH and serum from CSH rats were toxic in BIO, samples were dialyzed before assay. All samples were evaluated for PRL and GH by RIA as well. A significant decrease (P less than 0.05) in BIO serum PRL was evident in male rats 0.5 h after CSH; levels remained low for 24 h. Serum PRL by RIA was significantly depressed at 4 h but not at 0.5 h or 24 h. PRL in AP extracts was decreased (60-90%) at all times by BIO and RIA. Significant decreases of BIO- and RIA-detectable PRL were recorded in serum and tissues (AP and tumors) at 4 h in tumor rats. Sequentially bled (0.5-4 h) CSH-treated tumor-bearing rats showed 50% and 80% reductions in serum PRL at 1 and 4 h by both BIO and RIA. CSH had no effect on GH levels in sera and tissues of any animal studied at any time interval. Our results substantiate earlier reports on CSH-induced decreases in RIA-detectable PRL. They show that such changes cannot be attributed to assay effects alone, as significant decreases in circulating and stored PRL (both AP and tumor) were evident by BIO. Results with tissue extracts were the most dramatic. They suggest an action of CSH or a metabolic intermediate with stored PRL which reduces both extractable PRL and hormone release. Such an effect of CSH on PRL extraction has been suggested by others. Whatever the mechanism, it appears to be relatively specific, since GH cells were not affected.

Adaptation of islets of Langerhans to pregnancy: increased islet cell proliferation and insulin secretion correlates with the onset of placental lactogen secretion.

To elucidate the temporal profile of adaptive changes of the islets of Langerhans to the increased insulin demands of pregnancy, we have studied islet cell proliferation and insulin secretion during gestation in the rat. 5-Bromo-2'-deoxyuridine incorporation into dividing islet cells was significantly (P less than 0.05) increased over age-matched controls by day 10, rose continuously to a peak at day 14, and then returned to control levels by day 18. By day 20, cell division was significantly inhibited (P less than 0.05). The pattern of changes in insulin secretory profiles observed with perfused pancreata of pregnant animals was similar to that obtained for islet cell proliferation. Both the threshold of glucose-stimulated insulin secretion and the amount of above threshold insulin secretion began to diverge from controls by day 10. By day 12, the glucose-stimulation threshold was significantly decreased from 5.7 mM glucose to 3.3 mM (P less than 0.05), remained at this low level through day 15, and returned toward normal by day 20. Concomitant with the increased sensitivity of B cells to glucose, the above threshold insulin secretion was significantly increased by day 12 (P less than 0.05), peaked at day 15, and returned to control levels by day 20. This insulin secretory data demonstrates that the increased sensitivity of B cells to glucose is an important component of the adaptation of islets during pregnancy to the increased demand for insulin at physiological concentrations of plasma glucose. To correlate the above changes in islet cell proliferation and insulin secretion with levels of placental lactogen (PL), serum lactogenic hormone activity was measured by Nb2 lymphoma cell replication assays. This analysis revealed the expected biphasic pattern: a midpregnancy peak at day 12, followed by a nadir at day 14, and then continuously elevated levels until term. The bioassay data agreed with the known secretory profiles of rat (r) PL-I (midpregnancy) and rPL-II (late pregnancy). Our results provide the first systematic evaluation of changes in islet function during pregnancy in the rat. In addition, they provide evidence that rPL-I may be the critical hormonal signal which triggers the primary adaptive changes in islet function characteristic of pregnancy. The return to normal values of insulin secretion and inhibition of cell division observed at day 20 in the presence of high concentrations of rPL-II suggests that other inhibitory influences become dominant in the later stages of rat pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)

Parathyroid hormone stimulates the bone apposition rate independently of its resorptive action: differential effects of intermittent and continuous administration.

The deposition of mineralized bone matrix by differentiated osteoblasts was studied in rats in vivo by labeling the bone with three doses of tetracycline given at 48-h intervals. Only bone formation loci bearing all three tetracycline doses were measured, thus eliminating sites where bone formation was not continuous during the labeling period. Using this technique, the effects of intact bovine parathyroid hormone [bPTH-(1-84)] and of a synthetic amino-terminal fragment of human PTH [hPTH-(1-34)] were measured in thyroparathyroidectomized animals. bPTH-(1-84), administered sc, and hPTH-(1-34), administered iv, caused a dose-dependent increase in the bone apposition. Subcutaneous administration of hPTH-(1-34) in doses varying from 2.7-173.0 pmol/rat.day had no effect, probably due to the degradation of the hormone when administered this way. We also compared the effects of bPTH-(1-84) when administered by either daily sc injections or continuous infusion. Continuous infusion of bPTH-(1-84) resulted in an increased apposition rate. Using a morphometric technique, we also found an increase in both formation and resorption surfaces and a net decrease in the trabecular bone volume in this group. Daily injection of the hormone caused an increase in the bone apposition rate, accompanied by an increase in the formation surface without an increase in the resorption surface. This resulted in a net increase in trabecular bone volume. The results thus suggest that the resorptive effects of bPTH-(1-84) can be separated from the effects of the hormone on the apposition rate.

The effect of pinealectomy on seasonal changes in prolactin secretion in the white-tailed deer (Odocoileus virginianus borealis).

Surgery was performed on four buck and four doe 9-month-old white-tailed deer in March of 1978. Pinealectomy was performed on two deer of each sex, and the remaining animals received sham operations. At monthly intervals over the following year, baseline and TRH-stimulated (200 microgram/deer, iv bolus) serum PRL was measured over a 3-h period by RIA. Baseline PRL levels in sham-operated animals followed a circannual pattern, with peak levels occurring in June (74-237 ng/ml for does; 34-193 ng/ml for bucks) and lowest levels occurring in midwinter (0.41-0.44 ng/ml for does; 0.10-0.13 ng/ml for bucks). Pituitary responsivity to TRH followed the same patterns as that seen for basal PRL levels in sham-operated deer, with the highest peak serum PRL responses in June (198-568 ng/ml for does; 190-395 ng/ml for bucks) and the lowest peaks seen in midwinter (0.27-0.80 ng/ml for dose; 0.29-2.62 ng/ml for bucks). Pinealectomy appeared to abolish the circannual basal serum PRL rhythms in bucks, while this rhythm was maintained in does. Basal PRL levels in pinealectomized bucks ranged from 0.36-10.5 ng/ml, and basal levels in pinealectomized does ranged from 0.10-29.4 ng/ml. The greatest peak PRL response to TRH in pinealectomized deer was seen in August (41.2-93.4 ng/ml for does; 32.0-40.5 ng/ml for bucks), while the lowest peak response occurred in January (0.33-11.0 ng/ml for does; 0.50-17.0 ng/ml for bucks). Both sexes retained a degree of seasonality in their pituitary responsiveness to TRH, but the magnitude of the response in pinealectomized deer was greatly diminished in the summer months and increased in the winter months. Our results show that pinealectomy alters the naturally occurring photoperiod-linked seasonal profile of serum PRL in white-tailed deer and the associated pituitary responsiveness to TRH.

Effects of two treatment regimes with synthetic human parathyroid hormone fragment on bone formation and the tissue balance of trabecular bone in greyhounds.

The adult greyhound was found to be similar to adult man with respect to kinetic and histomorphometric indices of calcium metabolism. The relationship between trabecular bone tissue balance and the pattern of human PTH fragment 1-34 (hPTH 1-34) administration by daily injections or continuous sc infusions was investigated in this model and the results compared to those from a clinical trial of hPTH 1-34 in involutional osteoporosis (peptide administration by single daily injections). In the dogs, the daily injection regime elevated plasma levels of immunoreactive hPTH 1-34 for no more than 4 h/day. The greyhounds so treated showed significantly increased indices of bone formation (surface osteoid, plasma alkaline phosphatase activity, and skeletal accretion rate of calcium) and resorption (number of osteoclasts, resorption surfaces). Iliac trabecular bone volume increased significantly, as it did in the patients. The infusions did not significantly increase the trabecular bone volume or the 47Ca accretion rate, two parameters which increased in parallel in dogs and patients treated successfully by daily injections. The osteoclastic surfaces, however, were clearly increased by continuous infusions, while the increases in the osteoblastic surfaces were less statistically significant. Since hPTH 1-34 may inhibit osteogenesis in Friedenstein chambers, it is possible that the increased osteoblastic activity induced by the daily injection regime in trabecular bone is dependent on the noncontinuous nature of the PTH stimulus.

Dissociation of parathyroid hormone bioactivity and immunoreactivity in pseudohypoparathyroidism type I.

Circulating levels of parathyroid hormone (PTH) in six patients with pseudohypoparathyroidism type I (PSPI) have been measured by two immunoassays and by cytochemical bioassay and compared with measurements in normal subjects and patients with clinically defined hyper- and hypoparathyroidism. In all PSPI patients, the levels of immunoreactive PTH were in the hyperparathyroid range, whereas the bioactive levels were either in the normal or close to the normal range. In one patient from whom the dihydrotachysterol therapy was withdrawn, both immunoreactive and bioactive PTH concentrations increased. The finding that the PTH measured by RIA in these PSPI patients may have reduced biological activity may explain some of the clinical findings of hypoparathyroidism in this syndrome.

Radiation therapy for local-regional recurrent breast carcinoma.

One hundred seven women with recurrent breast carcinoma involving the chest wall and/or regional lymph node regions were treated with radiotherapy between 1970 and 1979. Local-regional tumor was the initial and only evidence of recurrent breast carcinoma in all cases. Forty-seven patients had their disease confined to the chest wall alone and sixty (56%) patients had chest wall involvement as some component of their local-regional recurrent disease. Within five years after the initial mastectomy, 80.5% of recurrences were manifested. All patients had radiotherapy to at least the site of involvement. Eighty-four patients (78.5%) had a complete response. The absolute 5-year survival of all patients following local-regional recurrence was 34.6%. Five year survival was 29% in those patients who had recurrence within 5 years of the original mastectomy. For those patients whose local-regional recurrence occurred after a 5-year disease-free interval, the subsequent 5-year survival was 57%. For patients with recurrence confined to the chest wall, subsequent 5-year survival was 48.9%. Patients who had supraclavicular involvement as part of their local-regional recurrence had only a 16.1% 5-year survival. The majority of patients developed distant metastasis. Twenty-two patients developed carcinoma of the contralateral breast following local-regional recurrence. Five year survival following local-regional recurrence was only 4.3% for patients whose initial treatment for their primary breast carcinoma was surgery and adjuvant chemotherapy. For those patients whose primary breast carcinoma was treated by surgery alone or surgery and post-operative radiotherapy, the 5-year survival following local-regional recurrence was over 40%.

Ten-year outcomes for pathologic node-positive patients treated in RTOG 75-06.

PURPOSE: This study was conducted to see what fraction of prostate cancer patients with biopsy-proven nodes are free of cancer 10 years after radiation treatment. METHODS AND MATERIALS: RTOG protocol #75-06 included 90 patients with biopsy-proven pelvic nodal involvement treated with radiation. They have been continuously follow-up since treatment. When feasible, current prostate-specific antigen (PSA) levels have been solicited from patients clinically cancer-free (no evidence of disease, NED) at 10 years, to confirm cure. RESULTS: The 10-year survival was 29%, the 10-year clinical NED survival 7%. PSA levels were obtained in 2 of 5 10-year clinical NED patients, they were both less than 0.8 ng/ml. The 2 proven cures were both clinical stage T-3, Gleason Score 6 and 8, and had 2 and 1 positive nodes, respectively. Multivariate analysis showed Gleason sum was significantly associated with clinical survival without disease. CONCLUSION: A small fraction of node-positive patients are cured at 10-year follow-up by radiation therapy (2 of 90 with PSA +3 of 90 by clinical endpoints). Innovative treatment programs should be directed at node-positive patients in an effort to improve the fraction cured.