Luteal phase ovarian oestrogen is not essential for implantation and maintenance of pregnancy from surrogate embryo transfer in the rhesus monkey

The aim of this study was to investigate whether luteal phaseovarian oestrogen is required for blastocyst implantation andpregnancy maintenance in the rhesus monkey. Preimplantationembryos were retrieved from naturally ovulated, mated embryodonor monkeys. In group I, developmentally normal, age- andstage-matched embryos were transferred to recipient monkeysshowing naturally synchronized ovulatory cycles. Immediatelyprior to embryo transfer, recipients were subjected to bilateralovariectomy, and following transfer they were treated with i.m.injections of either progesterone (group Ia, n= 4), or oestradiol+ progesterone (group Ib, n= 2). Recipient monkeys of groupIc (n= 4) were subjected to sham ovariectomy and vehicle injection.In group Ia, progesterone supplementation alone led to threepregnancies and live births. In group Ib, there was one livebirth. In the control group Ic, four transfers resulted in twolive births and one abortion on cycle day 58. Analysis of serumprogesterone and oestradiol profiles showed that oestradiolhad declined to undetectable levels within 3–5 days afterovariectomy in group Ia recipients, and the area under the curveof serum oestrogen concentrations during the peri-implantationperiod (days 10–20 after ovulation) were less (p< 0.001)in group Ia compared with group Ic. There were no changes inthe area under the curve among serum progesterone concentrationsin all the subgroups. In group II, long-term ovariectomizedembryo recipients (n= 4) were primed with oestradiol till cycleday 11 of simulated transfer cycle, and received progesteronetreatment from cycle day 10 till the end of the experiment.Of four transfers, live births were recorded in two cases, whilein one case abortion occurred on cycle day 66. Serum oestradiolconcentrations were undetectable during the presumptive peri-implantationperiod of pregnancy cycles in group II recipient monkeys. Noyes‘dating of endometrial samples collected from both groups ondays 5–7 after the oestrogen rise revealed that endometrialhistology synchronized well with those found during days 3–5after ovulation in normal menstrual cycle. We conclude thatluteal phase ovarian oestrogen is not essential for progesterone-dependentendometrial receptivity and response leading to implantationand pregnancy maintenance in the rhesus monkey.     

Epstein-Barr virus-associated gastric carcinoma in southern India: A comparison with a large-scale Japanese series

Epidemiological and clinicopathological features of Epstein-Barr virus (EBV) associated gastric carcinoma was compared in India and Japan, two countries differing markedly in gastric cancer incidence. Using in situ hybridization assay, the presence of EBV-encoded small RNA (EBER) was examined in 215, and 2,011 gastric cancer cases in Kerala, India, and Japan, respectively. Ten cases (5%), all males, in the Indian series were EBER-positive. This frequency was similar to that in the Japanese series (6.2%). As was the case with Japanese series, the EBV-associated gastric carcinoma in the Indian series was observed most frequently in the middle part of the stomach (1 in antrum, 4 in middle part, 2 in cardia, and 3 unknown), and, histologically, the diffuse type Lauren's classification (8 cases) was more common than the intestinal type (2 cases). Virus subtyping by PCR-RFLP revealed that all of the 10 EBV strains isolated from the EBER-positive Indian cases were subtype A, and wild-type F for Bam HI F region. In Bam HI I region, 8 cases were type C and the remaining 2 cases were type D. In either series, there was no significant difference in the frequency of tumors with p53 overexpression between EBER-positive and -negative cases. However, the proportion of cells with p53 overexpression in EBER-negative tumors was significantly higher than that in EBER-positive tumors regardless of histological type in both series. In conclusion, the frequency and major clinicopathological features of EBV-associated gastric carcinoma in south India were similar to those observed in Japanese series although gastric cancer incidence in these two countries differs markedly.     

Anti-nidatory effect of a single, early post-ovulatory administration of mifepristone (RU 486) in the rhesus monkey

The hypothesis that post-coital administration of mifepristone(RU 486) as a single dose in the early luteal phase can be aneffective anti-nidatory strategy was tested using the rhesusmonkey as the experimental model. Incidence of pregnancy, vaginalbleeding patterns, profiles of menstrual cyclicity and of serumlevels of progesterone and oestrogen were examined followingadministration of RU 486 as a single dose of 10 mg/kg and 2mg/kg body weight on the second day after ovulation. In controlmonkeys (group 1; n = 5) receiving the vehicle alone (benzylbenzoate: olive oil, 1: 4, v/v) there was a 60% pregnancy rate.Following s.c. administration of RU 486 at both doses, no pregnancywas recorded in a total of 33 treatment cycles in 12 monkeys.Five monkeys received RU 486 at 10 mg/kg s.c. (group 2) in threeconsecutive cycles. All animals had complete inhibition of implantation;in addition, the treatment cycle length was prolonged (P <0.001) due to an extension of the luteal phase. The subsequentfollicular phase was unaffected. Mild, premature vaginal bleedingduring the luteal phase was recorded in five treatment cycles,3–5 days after drug application. Though the serum profilesof progesterone and oestrogen in these monkeys showed markedindividual variations, there was a characteristic progesteronerebound about 18–20 days after drug administration. Monkeysin group 3 were given RU 486 at 2 mg/kg, s.c. either for threeconsecutive cycles (group 3a; n = 4) or for two consecutivecycles (group 3b; n = 3). Premature luteal phase vaginal bleedingoccurred only in four treatment cycles, within 2–6 dayspost-treatment. An increase in both the duration (P < 0.001)and degree (P < 0.001) of menstrual flow as compared withthe pre-treatment cycles was recorded in six treatment cyclesof three monkeys in group 3. These animals did not have prematureluteal phase vaginal bleeding. Collectively, 100% protectionagainst pregnancy with no change in the cycle length was obtainedin all seven monkeys in 18 treatment cycles. Analysis of pooleddata revealed that the subsequent follicular phase, as wellas the ovarian steroid hormone profiles of treatment cycleswere unaffected. Thus, a single application of RU 486 in theearly secretory phase appears to be a potential anti-implantationstrategy for intercepting pregnancy in the primate.     

Stability of antibody drug conjugate formulations evaluated using solid-state hydrogen-deuterium exchange mass spectrometry

Antibody drug conjugates (ADCs) have been at the forefront in cancer therapy due to their target specificity. All the FDA approved ADCs are developed in lyophilized form to minimize instability associated with the linker that connects the cytotoxic drug and the antibody during shipping and storage. We present here solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS) as a tool to analyze protein structure and matrix interactions for formulations of an ADC with and without commonly used excipients. We compared results of the ssHDX-MS with accelerated stability results using size-exclusion chromatography and determined that the former technique was able to successfully identify the destabilizing effects of mannitol and polysorbate 80. In comparison, Fourier-transform infrared spectroscopy results were inconclusive. The agreement between ssHDX-MS and stressed stability studies supports the potential of ssHDX-MS as a method of predicting relative stability of different formulations.     

Nondominant right coronary artery occlusion and ventricular tachyarrythmias.

It is usually considered that occlusion of a nondominant right coronary artery is not associated with significant consequences. We report two cases of nondominant right coronary artery occlusion that presented with sudden cardiac death. Timely intervention resulted in complete resolution of the ventricular arrhythmias. This highlights the need for greater vigilance in the recognition and treatment of these lesions.     

Admissions for CABG procedure in the elderly: was there a change in access to teaching hospitals after 1997?

The purpose of the study is to identify patient attributes associated with teaching hospital admissions in the elderly for coronary artery bypass graft (CABG), and to determine whether admission patterns in teaching hospitals by vulnerable subgroups of the elderly changed during 1997 to 2001, a period with significant changes in CABG admission patterns and financial situation faced by teaching hospitals. The study sample comprises elderly residents in two states, New York and Pennsylvania, and uses Healthcare Cost and Utilization Project State Inpatient data of the Agency for Health Care Research and Quality. Patient characteristics in major teaching hospitals are compared with those in rest of hospitals in a logistic regression framework using a pre-/postdesign, and controlling for county characteristics and resources, distance to hospitals, and hospital size and volume of procedures. Significant patient characteristics associated with a higher likelihood of admission to teaching hospitals included racial/ethnic minority status, transfer cases, Medicaid and private health maintenance organization insurance. A lower volume of CABG cases and an increased propensity to admit more complex cases characterized the admission patterns in teaching hospitals during 1997 to 2001. Although higher use of teaching hospitals by racial/ethnic minorities persisted, access for Medicaid patients disproportionately declined.     

Availability of essential medicines: A primary health care perspective

The present study was undertaken to assess the availability of the essential medicines to the people of Kunijarla, Khammam district, AP. This was done in two steps: 1) Comparison of PHC-EML (primary health care essential medicines list) with AP-EML (Andhra Pradesh essential medicines list) and NEML (National essential medicines list); and 2) Assessment of availability of the listed medicines in adequate quantity to meet the needs of people of kunijarla. Results showed that the PHC-EML is on par with AP-EML and NEML. The hospital has 100% availability of the listed medicines; however, there was a need for certain add-on drugs like phenytoin tablets, valproic acid, and activated charcoal since they were prescribed frequently and were not included in PHC-EML. All the drugs which were listed in PHC-EML were being used and none were considered as non-utility supply. Suggestions were given for the revision of PHC-EML according to the criteria for identifying medicines for inclusion in the revised list which was mentioned in NEML.     

Globin-coupled heme containing oxygen sensor soluble adenylate cyclase in Leishmania prevents cell death during hypoxia

Globin and adenylate cyclase play individually numerous crucial roles in eukaryotic organisms. Comparison of the amino acid sequences of globins and adenylate cyclase from prokaryotic to eukaryotic organisms suggests that they share an early common ancestor, even though these proteins execute different functions in these two kingdoms. The latest studies of biological signaling molecules in both prokaryotic and eukaryotic organisms have discovered a new class of heme-containing proteins that act as sensors. The protein of the globin family is still unknown in the trypanosomatid parasites, Trypanosome and Leishmania. In addition, globin-coupled heme containing adenylate cyclase is undescribed in the literature. Here we report a globin-coupled heme containing adenylate cyclase (HemAC-Lm) in the unicellular eukaryotic organism Leishmania. The protein exhibits spectral properties similar to neuroglobin and cytoglobin. Localization studies and activity measurements demonstrate that the protein is present in cytosol and oxygen directly stimulates adenylate cyclase activity in vivo and in vitro. Gene knockdown and overexpression studies suggest that O₂-dependent cAMP signaling via protein kinase A plays a fundamental role in cell survival through suppression of oxidative stress under hypoxia. In addition, the enzyme-dependent cAMP generation shows a stimulatory as well as inhibitory role in cell proliferation of Leishmania promastigotes during normoxia. Our work begins to clarify how O₂-dependent cAMP generation by adenylate cyclase is likely to function in cellular adaptability under various O₂ tensions.Human pathogen Leishmania promastigotes inhabit the midgut of a sandfly, where they are densely packed together and the environment of these promastigotes is then likely to become hypoxic or even anoxic (1). Although biologists have long known that Leishmania species can survive under limited oxygen (2), the underlying mechanism remains unclear.O₂, CO, and NO are physiologically indispensable but sometimes toxic to living organisms (3). Hence, the machinery for sensing these molecules and responding to them is vital for survival. Recently, a new class of the heme-containing sensor proteins has been discovered (4). These are different from well-known heme proteins, such as O₂ carriers (hemoglobin/myoglobin), oxygen activators (cytochrome P450/peroxidases), and mediators of electron transfer (cytochrome c/cytochrome b₅) (5, 6). Heme-based sensor proteins usually consist of an N-terminal heme-containing sensor domain and a C-terminal effector domain (7). The structures of heme-based sensor proteins are changed upon binding of O₂, CO, or NO gases with the heme domain. This structural change influences the effector domain for proper functioning.Cyclic AMP (cAMP) is a vital signaling molecule that controls diverse biological functions in many organisms, including virulence factors from a range of pathogens (8, 9). The second messenger cAMP is universally generated by adenylate cyclase (AC), which catalyzes the cyclization of ATP to cAMP. AC is regulated by various molecules including bicarbonate (10), calcium (11), and hormones (12). Although cAMP is found in the trypanosomatid parasite Leishmania, little is known about its function (13, 14).In this paper we report the heme containing AC from Leishmania major (HemAC-Lm). To understand the biochemical functions of this protein, we cloned, expressed, and characterized the HemAC-Lm protein. Evidence has been presented to show that a globin domain present at the N terminus of HemAC-Lm plays an important role in O₂ binding and controls the catalytic activity of the C-terminal AC domain. Our results provide evidence for the existence of an O₂-dependent cAMP regulation that is a prerequisite for decreasing oxidative stress under hypoxia.