GPR30 is necessary for estradiol-induced desensitization of 5-HT1A receptor signaling in the paraventricular nucleus of the rat hypothalamus.

Estrogen therapy used in combination with selective serotonin reuptake inhibitor (SSRI) treatment improves SSRI efficacy for the treatment of mood disorders. Desensitization of serotonin 1A (5-HT(1A)) receptors, which takes one to two weeks to develop in animals, is necessary for SSRI therapeutic efficacy. Estradiol modifies 5-HT(1A) receptor signaling and induces a partial desensitization in the paraventricular nucleus (PVN) of the rat within two days, but the mechanisms underlying this effect are currently unknown. The purpose of this study was to identify the estrogen receptor necessary for estradiol-induced 5-HT(1A) receptor desensitization. We previously showed that estrogen receptor ß is not necessary for 5-HT(1A) receptor desensitization and that selective activation of estrogen receptor GPR30 mimics the effects of estradiol in rat PVN. Here, we used a recombinant adenovirus containing GPR30 siRNAs to decrease GPR30 expression in the PVN. Reduction of GPR30 prevented estradiol-induced desensitization of 5-HT(1A) receptor as measured by hormonal responses to the selective 5-HT(1A) receptor agonist, (+)8-OH-DPAT. To determine the possible mechanisms underlying these effects, we investigated protein and mRNA levels of 5-HT(1A) receptor signaling components including 5-HT(1A) receptor, Gαz, and RGSz1. We found that two days of estradiol increased protein and mRNA expression of RGSz1, and decreased 5-HT(1A) receptor protein but increased 5-HT(1A) mRNA; GPR30 knockdown prevented the estradiol-induced changes in 5-HT(1A) receptor protein in the PVN. Taken together, these data demonstrate that GPR30 is necessary for estradiol-induced changes in the 5-HT(1A) receptor signaling pathway and desensitization of 5-HT(1A) receptor signaling.

Estradiol induces partial desensitization of serotonin 1A receptor signaling in the paraventricular nucleus of the hypothalamus and alters expression and interaction of RGSZ1 and Gαz.

Hyperactivity of hypothalamic-pituitary mediated hormone responses, such as to stimulation with a serotonin 1A (5-HT(1A)) receptor agonist, are a feature of depression which are normalized with clinical improvement during drug therapy. We previously reported that SSRIs induce desensitization of 5-HT(1A) receptor signaling in the paraventricular nucleus of the hypothalamus (PVN) while estradiol benzoate (EB) produces a more rapid, partial desensitization. In the current study, time course and dose-response experiments demonstrated that two once daily doses of EB is the minimum needed to induce the desensitization response as indicated by 5-HT(1A) receptor-stimulated release of oxytocin and that 10 µg/kg/day EB produces the maximal response, a partial desensitization of approximately 40%. The effects of two once daily injections of 10 µg/kg/day EB on Gαz and RGSZ1 proteins were examined as components of the 5-HT(1A) receptor signaling system, which mediates the release of oxytocin and adrenocorticotropic hormone. RGSZ1 appears to be a major target for EB-mediated responses in the 5-HT(1A) receptor signaling system. A 55 kD membrane-associate RGSZ1 protein was greatly increased in the PVN and rest of the hypothalamus and moderately increased in the dorsal hippocampus and amygdala after EB treatment as well as after an acute dose of a 5-HT(1A) receptor agonist. These results suggest that EB is a candidate for adjuvant therapy with SSRIs to hasten the therapeutic response and that RGSZ1 is a major target of EB therapy which could be explored as a target for novel therapeutic approaches for the treatment of depression.

Pulsatile intermittent intravenous insulin therapy for attenuation of retinopathy and nephropathy in type 1 diabetes mellitus.

Many hormones are secreted in a pulsatile fashion that is more efficient than continuous secretion when tested in vivo. A trial of multiple daily insulin doses with or without the addition of weekly pulsatile insulin infusion therapy was designed to determine if deterioration of renal and retinal function could be blunted. Sixty-five study subjects were evaluated prospectively in 7 centers. Thirty-six patients were randomly allocated to the infusion group and 29 to the standard therapy group. Mean serum creatinine was 1.6 mg/dL in both groups. Subjects were excluded if clearance was less than 30 mL/min. There were no significant differences between the groups with respect to age, duration of diabetes, sex distribution, glycohemoglobin, blood pressure, angiotensin-converting enzyme inhibitor use, proteinuria, or baseline diabetic retinopathy (DR) severity level (all eyes exhibited DR; 8 were deemed technically not amenable to evaluation). Progression of DR was noted in 31.6% of 57 patients (32.3% treated, 30.8% control; P = 1.0) with both eyes evaluable. For patients with 12 or more months of follow-up, 27.9% of 43 patients demonstrated progression of DR (32.0% treated, 22.2% control; P = .57). There were no significant differences between study groups with respect to progression or marked progression, nor was there any influence of duration of follow-up. Progression of DR was noted in 18.8% of 122 eyes that could be adequately evaluated (17.9% of 67 treated, 20% of 55 controls; P = .39). Serum creatinine increased to 1.7 mg/dL in the treatment group and to 1.9 mg/dL in the control group (P = .03). Statistically significant preservation of renal function by pulsatile insulin infusion was not matched by a statistically significant prevention of DR progression compared with standard diabetes care. Inadequate statistical power or duration of the study, or lack of further benefit of pulsatile insulin infusion on the retina in the presence of angiotensin-converting enzyme inhibition may be responsible.

Sharps injury reduction using Sharpsmart--a reusable sharps management system.

Sharps containers are associated with 11-13% of total sharps injuries (SI) yet have received little attention as a means of SI reduction. A newly developed reusable sharps containment system (Sharpsmart) was trialed in eight hospitals in three countries. The system was associated with an 86.8% reduction of container-related SI (CRSI) (P=0.012), a 25.7% reduction in non-CRSI (P=0.003), and a 32.6% reduction in total SI (P=0.002) compared with historical data. The study concludes that the Sharpsmart system is an effective engineered control in reducing SI.

Cloning and expressionanalysis of two cotton (Gossypium hirsutum L.) genes encodingcell wall proline-rich proteins.

Two cotton (Gossypium hirsutum L.) genes, ghprp1 and ghprp2, encoding cell wall proline-rich proteins (PRPs) have been cloned and characterized. The ghprpl gene has an open reading frame (ORF) that encodes a PRP of 299 amino acids (aa), whereas the ghprp2 gene contains an ORF that codes for a 310-aa PRP. The GhPRP1 has an 80% identity in aa sequence with that of GhPRP2. Like other plant cell wall PRPs, both cotton PRPs have a hydrophobic signal peptide at their N-termini, followed by repeating peptide units. Northern blot analyses showed that the ghprpl gene is predominantly expressed in the fiber during the elongation stage of fiber development. Reverse transcription (RT)-PCR analysis showed that ghprpl is expressed in both fiber and root tissues, whereas ghprp2 is in roots only. The ghprpl gene was shown to be present in the A1, A2, D1 and D5 genomes of Gossypium by PCR amplification, whereas the ghprp2 gene is only present in the A1 and A2 genomes. The ghprpl gene was over-expressed in the yeast Pichia pastoris, and the expressed GhPRP1 protein was used as an antigen to raise polyclonal antibodies (anti-GhPRP1). Western analysis using the anti-GhPRP1 probe detected a major protein band (50 kDa) in 5-31-day postanthesis (DPA) fibers. However, the 50 kDa protein was absent in other cotton tissues.

Effects of pulsatile intravenous insulin therapy on the progression of diabetic nephropathy.

The purpose of this study was to assess the effects of pulsatile intravenous insulin therapy (PIVIT) on the progression of diabetic nephropathy in patients with type 1 diabetes mellitus (DM). This 18-month multicenter, prospective, controlled study involved 49 type 1 DM patients with nephropathy who were following the Diabetes Control and Complications Trial (DCCT) intensive therapy (IT) regimen. Of these, 26 patients formed the control group (C), which continued on IT, while 23 patients formed the treatment group (T) and underwent, in addition to IT, weekly PIVIT. Blood pressure in all patients was maintained below 140/90 mm Hg on antihypertensive medication, preferentially using angiotensin-converting enzyme (ACE) inhibitors. All study patients were seen in the clinic weekly for 18 months, had monthly glycohemoglobin (HbA1c), and every 3 months, 24-hour urinary protein excretion and creatinine clearance (CrCl) determinations. The HbA1c levels declined from 8.61% +/- 0.33% to 7.68% +/- 0.31% (P = .0028) in the T group and from 9.13% +/- 0.36% to 8.19% +/- 0.33% (P = .0015) in the C group during the study period. CrCl declined significantly in both groups, as expected, but the rate of CrCl decline in the T group (2.21 +/- 1.62 mL/min/yr) was significantly less than in the C group (7.69 +/- 1.88 mL/min/yr, P = .0343). We conclude that when PIVIT is added to IT in type 1 DM patients with overt nephropathy, it appears to markedly reduce the progression of diabetic nephropathy. The effect appears independent of ACE inhibitor therapy, blood pressure, or glycemic control.

Cloning and promoter analysis of the cotton lipid transfer protein gene Ltp3(1).

A cotton Ltp3 gene and its 5' and 3' flanking regions have been cloned with a PCR-based genomic DNA walking method. The amplified 2.6 kb DNA fragment contains sequences corresponding to GH3 cDNA which has been shown to encode a lipid transfer protein (LTP3). The gene has an intron of 80 bp which is located in the region corresponding to the C-terminus of LTP3. The Ltp3 promoter was systematically analyzed in transgenic tobacco plants by employing the Escherichia coli beta-glucuronidase gene (GUS) as a reporter. The results of histochemical and fluorogenic GUS assays indicate that the 5' flanking region of the Ltp3 gene contains cis-elements conferring the trichome specific activity of Ltp3 promoter.

Cloning and characterization of a cotton lipid transfer protein gene specifically expressed in fiber cells.

A cotton genomic library was screened using a fiber-specific cDNA (GH3) encoding a lipid transfer protein (LTP). One genomic clone (1.7 kb DNA insert) containing the Ltp gene (Ltp6) was sequenced and characterized. The Ltp6 contains an open reading frame of 360 bp, which is interrupted by a single intron (136 bp) located in the region corresponding to the C-terminal of the protein. The derived amino-acid sequence of LTP6 is 64% homologous to that of GH3. Like the GH3 gene, the Ltp6 is specifically expressed in fiber cells in a temporal manner. However, its expression level is lower than that of GH3.

Differential expression of a lipid transfer protein gene in cotton fiber.

A full-length cDNA clone, GH3, has been isolated from a cotton fiber cDNA library using a differential screening method. The nucleotide and derived amino acid sequence data show that GH3 encodes a lipid transfer protein (LTP) of 120 amino acids. The presence of a transmembrane signal peptide at the N-terminal of the protein would suggest its possible outer cellular location in fiber cells. Northern analysis indicates that the GH3 gene is developmentally regulated.

Exaggerated radiation-induced fibrosis in patients with systemic sclerosis.

Four patients with stable systemic sclerosis and limited skin involvement received radiation for the treatment of solid malignant neoplasms. Following localized irradiation, each patient developed an exaggerated cutaneous and internal fibrotic reaction in the irradiated areas. The surface area of fibrosis extended beyond the radiation portals employed, and the fibrotic process was poorly responsive to antifibrotic therapy. Three of the patients died of complications caused by fibrous encasement of internal organs. The extent and severity of postradiation fibrosis in these patients was distinctly unusual. These observations suggest that patients with systemic sclerosis are particularly susceptible to developing excessive radiation-induced fibrosis.

Formylated peptides from cyanogen bromide digests identified by fast atom bombardment mass spectrometry.

Exposure of proteins to 70% formic acid during cyanogen bromide digestion can result in formation of artifact peaks during subsequent purification by HPLC and detection of [M + H + 28]+ ions during analysis by fast atom bombardment (FAB) mass spectrometry. Following cyanogen bromide digestion, peptides from equine heart cytochrome c, bacteriorhodopsin, bovine adrenal medulla dodecapeptide, and bovine adrenal peptide E were analyzed by positive ion FAB mass spectrometry. The cyanogen bromide peptides of cytochrome c and bacteriorhodopsin showed mixtures of formylated ions, [M + H + 28]+, and nonformylated ions, [M + H]+. Bovine adrenal medulla dodecapeptide and bovine adrenal peptide E were not formylated during digestion. Formylated peptides could be resolved from the corresponding nonformylated peptides using reversed-phase HPLC. Instability of the formylated peptides prevented localization of the adduct by Edman degradation. However, B/E-linked scanning during FAB mass spectrometric analysis with collisional activation of the [M + H + 28]+ ion of a cyanogen bromide peptide from cytochrome c suggested that formylation occurred at a threonine residue. On the basis of stability measurements in aqueous solution and analysis by FAB mass spectrometry, it was determined that serine and threonine residues are the most likely sites of esterification by formic acid during cyanogen bromide digestion of proteins. Furthermore, substitution of 70% trifluoroacetic acid for formic acid during cyanogen bromide digestion eliminated formylation and generated little or no trifluoroacetylation.

A random phase II study of mitoxantrone and cisplatin in patients with hepatocellular carcinoma. An ECOG study.

Of 86 patients entered in an Eastern Cooperative Oncology Group (ECOG) random Phase II study of mitoxantrone (DHAD) and cisplatin (DDP) in primary liver cancer, 69 were eligible. Nine of the 13 ineligible patients were excluded after a pathology review. Sixty-one percent of the patients were North American, and 39% were South African. The most common severe or the worst toxicity on DHAD was hematologic; and to DDP, hematologic and vomiting. Of the 69 eligible patients, 21 experienced severe, life-threatening or fatal toxic reactions. Two patients treated with DDP had partial responses. With a 95% confidence interval, the true response rate to DHAD was less than 8%, and to DDP, less than 17%. The median survival time was 14 weeks on both drugs. Assuming a proportional hazards model, factors that are significantly associated with survival are patient performance status, the presence of the symptoms, raised bilirubin and hepatomegaly, and clinical evidence of cirrhosis. Any differences between survival rates for South African and North American patients were largely explainable by these factors.

Actual versus ideal weight in the calculation of surface area: effects on dose of 11 chemotherapy agents.

This study of 2382 breast, 182 rectal, 817 colon, and 351 lung cancer patients treated with combination chemotherapy on eight phase III Eastern Cooperative Oncology Group protocols indicates that 69% would receive a higher dose of at least one drug if surface area were calculated from actual weight rather than from the minimum of actual and ideal weight. Forty-eight percent of the patients would have at least a 10% increase in drug dose based on actual weight. Only on the premenopausal adjuvant breast cancer protocol and among women on the rectal adjuvant study do the differences in dose based on actual rather than ideal weight increase significantly with age. On the postmenopausal adjuvant breast study and on the lung cancer study, the differences in dose decrease significantly with age. For all age decades and both sexes within each protocol, the mean differences between dose based on actual and dose based on ideal weights were on the same order as the rounding factors for the 11 drugs studied. From the literature on the effect of doses of common chemotherapies on leukopenia, it appears that the percent of hematologic toxicity would not be raised to unacceptable levels by using actual weight to set doses.

Prospectively randomized clinical trial of three intensive chemotherapy regimens for the treatment of advanced unfavorable histology non-Hodgkin's lymphoma.

Three hundred thirty-two eligible patients with advanced (Ann Arbor stage III or IV) non-Hodgkin's lymphoma of aggressive histologic subtype (Rappaport classification diffuse histiocytic [DH], diffuse poorly differentiated lymphocytic [DPDL], diffuse mixed [DM], or diffuse undifferentiated [DU]) were randomly assigned to receive induction chemotherapy with one of three intensive regimens in a clinical trial conducted by the Eastern Cooperative Oncology Group (ECOG) between 1978 and 1983. Chemotherapy regimens consisted of cyclophosphamide, vincristine, prednisone, and doxorubicin (Adriamycin; Adria Laboratories, Columbus, OH) (COPA) administered in 3-week cycles; cyclophosphamide plus doxorubicin plus prednisone beginning day 1, with vincristine plus bleomycin day 15 of each 3-week cycle (COPA + Bleo); or cyclophosphamide plus doxorubicin plus procarbazine beginning day 1, and bleomycin plus vincristine plus prednisone beginning day 15 of each 4-week cycle (CAP-BOP). The median patient follow-up from study entry for patients still alive is 5 years. The three regimens were not significantly different with respect to complete response (CR) rates (43% to 46%), time to progression of malignant disease (median, 1.0 to 1.7 years), or survival (5-year survival, 34% to 45%), although duration of complete remission appeared to be shorter in patients receiving COPA (P = .03). COPA + Bleo and CAP-BOP were significantly more toxic than the COPA regimen. This study did not demonstrate any substantial therapeutic advantage associated with the addition of a fifth or sixth chemotherapy drug, or with treatment administered on a more frequent administration schedule, compared with the COPA regimen in this population of patients with advanced diffuse non-Hodgkin's lymphoma. The relatively small proportion of long-term disease-free survivors treated with COPA underscores the need for prospective clinical trials of new and more effective treatments for patients with these potentially curable tumors.

Randomized comparison of three adriamycin regimens for metastatic soft tissue sarcomas.

This study addressed two major questions regarding therapeutic use of Adriamycin ([Adr] Adria Laboratories, Columbus, OH) in adult soft tissue sarcomas: the influence of dosing schedule and the value of adding imidazole carboxamide (DTIC) to Adr. Patients with objectively measurable metastatic soft tissue sarcomas were randomized to Adr 70 mg/m2 intravenously (IV) day 1 and every 3 weeks (94 patients); Adr 20 mg/m2 IV day 1, 2, and 3, and 15 mg/m2 IV day 8 and weekly thereafter (89 patients); and Adr 60 mg/m2 IV day 1 and DTIC 250 mg/m2 days 1 to 5, repeated every 3 weeks (92 patients). The regimens using Adr as a single agent resulted in an equivalent response frequency (18% and 16%) and survival (median, 8.0 and 8.4 months). DTIC significantly increased (P less than .02) the overall response frequency of Adr to 30%. However, DTIC did not influence survival (median, 8.0 months) or increase the number of complete responses. The toxicities of the two single-agent regimens differed: Adr weekly resulted in more stomatitis (P = .09) and less hematologic toxicity (P less than .05). DTIC resulted in substantially increased toxicity, primarily gastrointestinal (P less than .002); overall, 98% of patients receiving Adr-DTIC experienced moderate or worse toxicity. To decrease the potential for error in interpretation of treatment results, histopathological confirmation of diagnosis was undertaken by a panel of reference pathologists; pathology slides were submitted on 97% of entered patients. The on-study clinical diagnosis was affirmed in 199 of 316 patients (63%) with a final review. In 23% of patients, the panel agreed with the diagnosis of soft tissue sarcoma, but not with the type. In 14%, the panel concluded that a diagnosis of mesenchymal malignancy could not be affirmed. Final treatment results were based on the 275 pathologically confirmed, eligible patients. The most common histological subtype entered was leiomyosarcoma (99 patients). The response to Adr-DTIC of this subtype was higher (44%) than that of any other subtype. However, this difference alone was not responsible for the overall superiority of the combination. This confirmed that the combination of DTIC plus Adr adds to the response rate of Adr alone in soft tissue sarcomas. Whether the increased response frequency, without an impact on survival, is worth the significantly greater toxicity remains a subjective judgement that must be made within the context of the individual patient.

Randomized comparison of cyclophosphamide, imidazole carboxamide, and adriamycin versus cyclophosphamide and adriamycin in patients with advanced stage malignant mesothelioma: a Sarcoma Intergroup Study.

In 1980, a consensus chemotherapy intergroup study for advanced malignant mesothelioma was initiated based on a collaborative agreement among the Eastern Cooperative Oncology Group (ECOG), the Southwest Oncology Group (SWOG), and the Southeastern Cancer Study Group (SECSG). The purpose of the study was to evaluate cyclophosphamide (500 mg/m2 day 1), imidazole carboxamide (250 mg/m2 days 1 through 5), and doxorubicin (Adriamycin; Adria Laboratories, Columbus, OH) (50 mg/m2 day 1) v cyclophosphamide (500 mg/m2) and doxorubicin (50 mg/m2) in a randomized prospective clinical trial involving 76 fully evaluable patients with advanced stages II to IV malignant mesothelioma. A total of nine responses (12%) were documented, including three complete and six partial responses. There was no significant difference in response duration or survival between treatment arms. Leukopenia (greater than 2,000/microL) was observed in 46% of patients treated with the three-drug combination and 38% of patients receiving the two-drug combination. The variables of performance status 0-1 and the absence of prior chemotherapy/radiotherapy were significant with respect to favorable impact on survival. We conclude, based on the minimal benefit observed, that the combination of cyclophosphamide and doxorubicin with or without imidazole carboxamide does not warrant further investigation in patients with advanced-stage malignant mesothelioma.

Phase II evaluation of dibromodulcitol and actinomycin D, hydroxyurea, and cyclophosphamide in previously untreated patients with malignant melanoma.

In this Eastern Cooperative Oncology Group (ECOG) phase II study, dibromodulcitol (DBD) and a combination of actinomycin D, hydroxyurea, and cyclophosphamide (AHC) were compared with methyl-CCNU, the current ECOG standard, in patients who had received no prior chemotherapy for disseminated malignant melanoma. The response rates were 6% (3/50) for AHC, 9% (3/34) for DBD, and 14% (7/49) for methyl-CCNU. Median survival times were 4, 5, and 6 months, respectively. Neither regimen appears to offer any advantage over methyl-CCNU as front-line therapy for patients with disseminated melanoma.

Megestrol acetate v tamoxifen in advanced breast cancer: correlation of hormone receptors and response.

This report describes the preliminary results from a randomized study comparing megestrol acetate with tamoxifen in the treatment of postmenopausal women with advanced breast cancer, correlating estrogen receptors (ER) and progesterone receptor (PgR) status with response. Patients received megestrol acetate (40 mg) orally four times daily or tamoxifen (10 mg) orally twice daily. If the initial therapy failed, patients were crossed over to the alternate treatment. Of 197 patients entered in the study, 190 were considered evaluable. The overall response rates were 35% with megestrol acetate and 42% with tamoxifen. Twenty-three percent (7/30) of patients responded to megestrol acetate after being crossed over from tamoxifen, while 22% (6/27) responded to tamoxifen after being crossed over from megestrol acetate. Response did not correlate significantly with combined receptor (ie, ER plus PgR) levels. A significant trend was seen between ER level and response only in the tamoxifen group. There was no association between PgR levels and response for either tamoxifen or megestrol acetate.

A randomized trial of the four most active regimens for metastatic non-small-cell lung cancer.

Between October 1981 and June 1983, the Eastern Cooperative Oncology Group (ECOG) conducted a prospectively randomized trial (EST 1581) of the four most active chemotherapy regimens for metastatic non-small-cell lung cancer (NSCLC). Four hundred eighty-six good performance status patients (PS 0 or 1; 81%) were randomized to receive cyclophosphamide, doxorubicin, methotrexate, and procarbazine (CAMP); mitomycin, vinblastine, and cisplatin (MVP); etoposide and cisplatin (VP-P); or vindesine and cisplatin (VDA-P). All regimens were administered in the doses and schedules originally reported. Complete response (CR) plus partial response (PR) rates for the four regimens were CAMP, 17%; MVP, 31%; VP-P, 20%; and VDA-P, 25%. The response rate for MVP was significantly higher in patients with squamous and adenocarcinoma histologies, but there was no impact on median survival (overall, 24.5 weeks). The duration of response did not differ by treatment as previously suggested for VDA-P. There were 15 CRs (CAMP, one; MVP, six; VP-P, two; VDA-P, six), and 12 patients have survived more than 2 years. Toxicity was significant with 20 treatment-related deaths. CAMP was significantly less toxic than the other regimens (P less than .001). VDA-P demonstrated significantly more life-threatening (seven) and lethal (three) episodes of nephrotoxicity (P less than .001) despite an aggressive hydration program that in itself caused significant morbidity. Analysis of the toxicity data showed, however, that most of the severe toxicity occurred in the 19% of patients who were initially PS 2, suggesting that they are not appropriate candidates for trials of new agents or combinations. None of these regimens can be recommended as a standard therapy for metastatic NSCLC.