Conventional surgery in colon cancer with comparison to complete mesocolic excision and central vascular ligation: initial experience in a tertiary centre.

INTRODUCTION: The complete mesocolic excision (CME) and central vascular ligation (CVL) is an advanced surgical technique used to treat colon cancer. It combines the removal of the affected portion of the colon and surrounding lymph nodes with an improved method of controlling the vascular supply to the tumour. MATERIALS AND METHODS: A retrospective study of patients with colon cancer underwent right hemicolectomy (either CME and CVL or conventional method) were operated by colorectal surgeons in a tertiary centre in Kuala Lumpur from 2018 to 2020. We review the data to compare the oncological, pathological and surgical outcomes of both techniques. Categorical variables were presented as frequencies and percentages. Continuous variables were compared using an independent t-test or Mann-Whitney Rank U test. The chi-square test was used to determine the association between categorical variables and mortality. Statistical analysis was conducted with IBM SPSS Statistics 25.0, and statistical significance was set at p<0.05. RESULTS: A total of 30 patients (CME and CVL=15 or conventional colectomies=15) were included in this study with mean age of 65 years. There was no statistical difference between the mean age of the two groups (p=0.355). Most of the patients were Malays (46.7%) followed by Chinese (43.3 %) and Indians (10.0%). The mean (SD) = 19 (9) number of lymph nodes harvested is more in CME and CVL groups which however is not statistically significant compared to the mean (SD) = 16 (9), number of lymph nodes in conventional colectomies. The duration of surgery is longer in CME and CVL groups (214 minutes) compared to conventional colectomies (188 minutes) but with no significant statistical difference. Most of the perioperative complications were similar in both groups with no significant statistical differences. CONCLUSION: CME and CVL are not inferior to conventional surgery in colon surgery in a tertiary centre. It should be considered since the advantages such as lymph node yield and median recurrence free survival are better with similar perioperative morbidity.

Implementing primary eye care in private practises in Malaysia: the challenges faced by optometrists.

OBJECTIVE: In Malaysia, optometrists' role in the private sector is limited compared to their counterparts elsewhere. Primary eye care (PEC) is still not widely offered in private practises despite its demand to reduce the public's eye morbidity. This study aims to explore the challenges perceived by the private sector optometrists in implementing PEC in Malaysia. MATERIALS AND METHODS: In-depth interview using semistructured open-ended questions were designed to explore the challenges of implementing PEC. Fifteen private optometrists across Malaysian were interviewed via purposive sampling until the data were saturated. The interviews were audio-recorded, transcribed and analysed. RESULTS: Four major themes emerged: working environment, support and recognition, self-sufficiency and customer influence. The first major theme identified a lack of time and equipment in the workplace as a barrier to PEC implementation. The second major theme acknowledges the lack of support and recognition for PEC practise from financial bodies, the government, Malaysian Optical Council (MOC) and other eye professionals. Meanwhile, some practising optometrists faced significant challenges due to their lack of self-sufficiency regarding skills, knowledge and confidence. The final major theme, customer influence, reflects the customer's role in shaping eye care delivery through their perception and acceptance of PEC. CONCLUSION: Each of the issues identified played a significant impact in impeding PEC implementation in Malaysia. This study is the first step toward developing tailored interventions to improve eye care delivery in Malaysia.

Decidualization of the canine uterus: From early until late gestational in vivo morphological observations, and functional characterization of immortalized canine uterine stromal cell lines.

The apparent lack of classical mechanisms for maternal recognition of pregnancy is one of the most intriguing features of canine reproduction. Consequently, similar levels of circulating luteal steroids are observed in pregnant and non-pregnant dogs. However, the early pre-implantation canine embryo locally modulates uterine responses to its presence, facilitating the successful onset of pregnancy. As a part of this interaction, the canine uterus undergoes a species-specific decidualization. Maternal stroma-derived decidual cells develop, the only cells of the canine placenta expressing progesterone receptor (PGR). There exists an acute need for an in vitro stable cell line model for canine decidualization. Therefore, herein our goal was to establish, immortalize and characterize such a cell line. We immortalized three monolayer dog uterine stromal (DUS) cell lines by stably transfecting them with SV40Tag oncogene. Cells retained their mesenchymal character for over 30 passages, as evidenced by VIMENTIN staining. Genomic incorporation of the SV40Tag protein was confirmed by immunofluorescence and Western blot analyses. Cells submitted to a classical in vitro decidualization protocol (N6,2'-O-dibutyryladenosine-3',5'-cyclic monophosphate) revealed upregulated gene levels of selected major decidualization markers (e.g. PRLR, PGR, IGF1, PTGES). Additionally, the basic decidualization capability of PGE2 was demonstrated, revealing increased levels of, for example, PGR and PRLR gene expression, thereby implying its involvement in the progesterone-dependent decidualization in the canine uterus. In summary, our in vitro model with immortalized DUS cell line could serve as an ideal and unique model to study the underlying molecular and endocrine mechanisms of canine decidualization.

Room temperature ammonia sensing using tapered multimode fiber coated with polyaniline nanofibers.

We demonstrate an ammonia sensor composed of a tapered multimode fiber coated with polyaniline nanofibers that operates at room temperature (26°C). The optical properties of the polyaniline layer changes when it is exposed to ammonia, leading to a change in the absorption of evanescent field. The fiber sensor was tested by exposing it to ammonia at different concentrations and the absorbance is measured using a spectrophotometer system. Measured response and recovery times are about 2.27 minutes and 9.73 minutes, respectively. The sensor sensitivity can be controlled by adjusting the tapered fiber diameter and the highest sensitivity is achieved when the diameter is reduced to 20 µm.

Choroidal osteoma - case reports.

Choroidal osteoma is a rare disease. In this article four case histories were described. All were female and young patient. One patient had bilateral and other three had unilateral involvement. They had no family history. One patient reported at eye department in Bangabandhu Sheikh Mujib Medical University (BSMMU) and the other three patients reported in Bangladesh Eye Hospital. Choroidal osteoma is a benign tumor. It is diagnosed by fundoscopy, ocular B-scan ultrasonography, x-ray orbit, FFA, OCT and CT-scan of orbit. Most patients do not require treatment. Hemorrhage on the lesion suggests the presence of sub-retinal neovascularization which are typically treated with laser or intra-vitreal anti-VEGF.

Ototoxicity screening of patients treated with streptomycin using distortion product otoacoustic emissions.

OBJECTIVE: Pure tone audiometry (PTA) is currently widely used to monitor ototoxicity, but this method is time-consuming. Here we validate distortion product otoacoustic emission (DPOAE) as an instrument for early detection of ototoxicity. METHODS: A cohort study was performed on newly diagnosed tuberculosis patients who were treated with streptomycin. The patients underwent hearing assessment using conventional PTA and high-frequency DPOAE (8, 9 and 10 kHz) on days 0, 7, 14, 28 and 56 of streptomycin treatment. Detection of ototoxicity according to the duration of streptomycin treatment was compared between DPOAE and PTA. RESULTS: Of 96 newly diagnosed patients treated with streptomycin, 50 completed the study. During the treatment period, 62.5% of the patients had vertigo, while 37.5% complained of tinnitus. DPOAE detected ototoxicity in 47.7% of the cases at day 7, 66.0% at day 14, 70.0% at day 28 and 77.1% at day 56 of streptomycin treatment. The higher frequencies were affected more by ototoxicity, with significant differences at 8 vs. 9 kHz on all testing days and at 9 vs. 10 kHz except on days 7 and 56 (p < 0.001). Hearing loss was detected by PTA in 2.3% of patients on day 7, in 10.6% on day 14, in 22.0% on day 48 and in 29.2% on day 56. CONCLUSION: DPOAE is a sensitive tool that can detect early changes in the cochlea due to ototoxicity. Use of DPOAE rather than PTA to screen for ototoxicity could reduce screening time and would allow clinical monitoring of more patients.

Mitigating mutual coupling effects on circular polarization for improved bandwidth in MIMO systems: A novel approach.

An improved mutual coupling compensation in circularly polarized (CP) multi-input multi-output (MIMO) dielectric resonator antenna (DRA) is presented in this paper. Using trimming approach, the mutual coupling (MC) between closely spaced DRA units at 0.3λ has been significantly reduced while axial ratio performance has been maintained. Mutual coupling reduction is obtained by trimming the DRA to ensure low mutual coupling below -20dB. The exclusive features of the proposed MIMO DRA include wide impedance matching bandwidth (BW), triple band circular polarization, and suppressed MC between the radiating elements. The impedance bandwidth matches perfectly with a triple band's 3 dB axial ratio (AR). It is designed with characteristic mode analysis with good agreement of the measurement that has been obtained. Using the probe feed method, the DRA and patch strip are coupled together to allow bandwidth widening of the pro-posed DRA. An impedance bandwidth of 34% at a lower frequency to around 2% at a higher frequency was achieved in all resonance frequencies. Thus, we refer to our newly designed DRA as a proposed method for effectively reducing the mutual coupling between DRAs. Additionally, the 3 dB AR bandwidth matched at 3.3 GHz, 4.6 GHz, and 6.3 GHz with a percentage of 11.66%, 3.04%, and 2.22% obtained at the three different frequencies. Note that the proposed DRA exhibits low mutual coupling (below -20 dB) at the targeted frequencies, which is suitable for better signal reception for MIMO applications. By computing, the metrics envelop correlation coefficient, diversity gain, channel capacity loss, and total active reflection coefficient, the MIMO performance of the proposed antenna is verified. The experiments show a close result between simulated and computed validation of the proposed DRA.

Development of a mental script for the mental practice of micro suturing: a methodological approach.

INTRODUCTION: Motor imagery and mental practice are important for the acquisition and mastery of surgical skills. The success of this technique relies on the use of a well-developed mental script. In this study, we shared how we developed a mental script for basic micro suturing training by using a low-fidelity rubber glove model. METHODS: This study applied the design and development research framework. Five expert surgeons developed a mental script by performing a cognitive walkthrough to repair a vertical opening in a rubber glove model, followed by hierarchical task analysis. A draft script was created, and its face and content validity assessed with a checking-back process. Twenty-eight surgeons used the Mental Imagery Questionnaire (MIQ) to assess the validity of the final script. RESULTS: The process of developing the mental script is detailed. The assessment by the expert panel showed the mental script had good face and content validity. The mean overall MIQ score was 5.2±1.1 (standard deviation), demonstrating the validity of generating mental imagery from the mental script developed in this study for micro suturing in the rubber glove model. CONCLUSION: The methodological approach described in this study is based on a design and development research framework to teach surgical skills. This model is inexpensive and easily accessible, addressing the challenges of reduced opportunities to practise surgical skills. However, although motor skills are important, the surgeon's other non-technical expertise is not addressed with this model. Thus, this model should act as one surgical training approach, but not replace it.

Integrating instructional design principles into surgical skills training models: an innovative approach.

OBJECTIVE: Surgical training programmes are evolving from time-based to competency-based schedules, which define expected learning outcomes in surgical knowledge, clinical and technical skills according to training levels. This article aims to review current models in surgical skills acquisition and to propose an integrative process-driven, outcomes-based model for surgical skills acquisition and mastery. DESIGN: A literature review was conducted on the theories of motor skills acquisition using PubMed, Web of Science and Google Scholar from 2010 to February 2020. The review was limited to theories and models on surgical skills acquisition and mastery. Four models of surgical skills acquisition were included: Fitts and Posner's three-stage model of motor skills acquisition, Bandura's social learning theory, Ericsson's deliberate practice model and Jeannerod's motor simulation theory. These models are deficient in that there is no universally accessible opportunity to practise the surgical procedure outside of the operating theatre and without access to physical simulators. RESULTS: We propose an innovative model that allows deliberate practice of the procedure without the need for expensive physical simulators, and provides an on-demand, self-directed practice by the trainees to achieve the level of mastery. This new model, which incorporates motor imagery and mental practice, augmented by deliberate practice, will provide an alternative training path for expert performance in surgical procedures. CONCLUSIONS: The innovative model provides a solution to the reduced opportunity for practice by surgical trainees to achieve mastery in surgical motor skills.

Nanoparticles carrying fingolimod and methotrexate enables targeted induction of apoptosis and immobilization of invasive thyroid cancer.

Metastatic tumors are the main cause of cancer-related death, as the invading cancer cells disrupt normal functions of distant organs and are nearly impossible to eradicate by traditional cancer therapeutics. This is of special concern when the cancer has created multiple metastases and extensive surgery would be too dangerous to execute. Therefore, combination chemotherapy is often the selected treatment form. However, drug cocktails often have severe adverse effects on healthy cells, whereby the development of targeted drug delivery could minimize side-effects of drugs and increase the efficacy of the combination therapy. In this study, we utilized the folate antagonist methotrexate (MTX) as targeting ligand conjugated onto mesoporous silica nanoparticles (MSNs) for selective eradication of folate receptor-expressing invasive thyroid cancer cells. The MSNs was subsequently loaded with the drug fingolimod (FTY720), which has previously been shown to efficiently inhibit proliferation and invasion of aggressive thyroid cancer cells. To assess the efficiency of our carrier system, comprehensive in vitro methods were employed; including flow cytometry, confocal microscopy, viability assays, invasion assay, and label-free imaging techniques. The in vitro results show that MTX-conjugated and FTY720-loaded MSNs potently attenuated both the proliferation and invasion of the cancerous thyroid cells while keeping the off-target effects in normal thyroid cells reasonably low. For a more physiologically relevant in vivo approach we utilized the chick chorioallantoic membrane (CAM) assay, showing decreased invasive behavior of the thyroid derived xenografts and an increased necrotic phenotype compared to tumors that received the free drug cocktail. Thus, the developed multidrug-loaded MSNs effectively induced apoptosis and immobilization of invasive thyroid cancer cells, and could potentially be used as a carrier system for targeted drug delivery for the treatment of diverse forms of aggressive cancers that expresses folate receptors.

Study on the physical and chemical composition of agro wastes for the production of 5-hydroxymethylfurfural.

Treated sludge, goat manure, sugarcane bagasse, empty fruit bunches of oil palm (EFBP) and dry leaves are agro wastes that have high potential for use as feedstocks for the production of 5-hydroxymethylfurfural (5-HMF). The focus of this study is to investigate the production of 5-HMF from agro wastes via co-hydrothermal (CHT) treatment and extraction. Present study include examine on agro waste's physical and chemical properties and also their thermal degradation behaviour. The analysis of the bio-oil products is conducted by FTIR and GC-MS. Co-hydrothermal experiments were conducted at a temperature of 300°C with an experimental time of 15min, followed by alcohol extraction. Highest carbon and hydrogen content are 45.94% and 6.49% (dry leaves) with maximum high heating value 18.39MJ/kg (dry leaves) and fix carbon value 6.60 (goat manure). Through CHT about 39% 5-HMF, 22.97% carboxylic acids, 0.97% of aromatic and 0.73% aldehyde obtained.

Use of hecate-chorionic gonadotropin beta conjugate in therapy of lutenizing hormone receptor expressing gonadal somatic cell tumors.

Improvement of cancer treatment is a major challenge of medical research. Despite the immense efforts made in the improvement of diagnosis and treatment, cancer remains a major concern and cause of morbidity and mortality. Most of the modern anti-neoplastic therapies have severe side effects, and tumor cells often develop drug resistance. There is promise in the new generation of treatments (gene therapy, immunotherapy, vaccines, etc.) that are under development, but the efficacies and side effects of such therapies have so far been disappointing. Receptor-based therapies are not new, but many normal cells also present the same receptors reducing the specificity of such approaches. Several lytic peptides have been investigated because of they appear to kill cancer cells due to changes of their membrane potential. Thus, linking receptor-specific ligands to lytic peptides is expected to augment the specificity of targeting and decrease the toxicity of lytic peptides on normal cells. One such polypeptide is hecate (an analogue to the bee venom main component, melittin) that preferentially kills cancer cells at low doses. When this peptide is fused with the 81-95 amino acid fragment of chorionic gonadotropin-beta (CGbeta) subunit (hecate-CGbeta), it targets cells expressing luteinizing hormone receptor (LHR), even at very low doses, or when LHR is expressed at low level. Our recent data showed that this peptide conjugate is efficient in destroying LHR-positive cells in xenografts and more importantly in transgenic mouse models developing LHR-positive somatic cell tumors in gonads. The mechanism of action of hecate-CGbeta after binding to LHR is destruction of cell membranes resulting in rapid cell death by necrosis with minimal side effects. This review summarizes our findings on the action of this novel peptide and considers the future potential of this family of targeting peptides in the treatment of neoplasias.

Maxillofacial trauma with emphasis on soft-tissue injuries in Malaysia.

Soft-tissue injuries with or without facial bone involvement are the most common presentation following maxillofacial trauma. The objective of this study was to look at the distribution, pattern and type of soft-tissue injury in relation to aetiology. Records of patients over a period of 5 years (1998-2002), who sustained maxillofacial injuries and were treated at Kajang Hospital, a secondary referral hospital, were reviewed. Out of 313 patients with maxillofacial injuries, 295 patients sustained soft-tissue injuries. Males (79%) between 21 and 30 years old (34%) were the majority of patients. Road-traffic accident was the main cause of soft-tissue injuries (75%) with motorcycle accident being the most frequent (40%). The upper lips (23%) and the lower lips (18%) were the most common extraoral site involved, while the labial mucosa and sulcular areas, both accounting for 21%, were the most common intraoral sites. Stringent road-traffic regulations should be practiced in developing countries, as morbidity arising from road-traffic accidents poses a national economic and social problem.

The synthetic molecules YK51 and YK73 attenuate replication of dengue virus serotype 2.

Dengue virus infection has been posing alarming economic and social burden on affected nations. It is estimated that 50-100 million dengue infections occur annually with over 2.5 billion people at risk for endemic transmission. In the effort to develop effective antiviral agents, we previously reported potential antiviral activities from selected array of natural products and compounds against dengue virus serotype 2 (DV2). In this study, we report the synthesis of two efficacious novel compounds, YK51 and YK73, and their activities against DV2 replication. Both compounds were chemically synthesised from nicotinic acid using a modified method for the synthesis of dihydropyridine. The products were tested with cell-based assays against DV2 followed by a serine protease assay. As a result, both YK51 and YK73 exhibited intriguing antiviral properties with EC50 of 3.2 and 2.4 µM, respectively. In addition, YK51 and YK73 were found to attenuate the synthesis of intracellular viral RNA and protect the switching of non-classic mechanism of protein translation. These compounds demonstrated inhibitory properties toward the activity of DV2 serine protease in a dose dependent manner. These findings demonstrate that both YK51 and YK73 serve as DV2 serine protease inhibitors that abrogate viral RNA synthesis and translation. Further investigation on these compounds to corroborate its therapeutic properties towards dengue is warranted.

Molecular dynamics of luteinizing hormone receptors on rat luteal cells.

To better understand the in situ organization of the luteinizing hormone receptor on rat luteal cells, we have examined the molecular motions of this receptor following binding of ovine luteinizing hormone (oLH) or human chorionic gonadotropin (hCG). Fluorescence photobleaching recovery (FPR) measurements of LH receptor lateral diffusion were performed using tetramethylrhodamine isothiocyanate (TRITC)-derivatized oLH or hCG as a probe. These experiments indicate that TRITC-oLH occupied LH receptors on luteal cells obtained from superovulated female rats have a lateral diffusion coefficient D of (1.7 +/- 0.6).10(-10) cm2s-1 at 27 degrees C with fluorescence recovery after photobleaching of 46 +/- 5%. In similar experiments, binding of TRITC-hCG caused a significant decrease in LH receptor lateral diffusion; fluorescence recovery after photobleaching was less than 20%. To determine whether hCG-occupied receptors might exist in large aggregates, we measured the rotational correlation times (RCT) of hCG and oLH bound to the LH receptor on intact cells using single cell polarized fluorescence depletion (PFD). At 4 degrees C, LH receptors occupied by eosin isothiocyanate (EITC)-hCG exhibited a slower RCT (64 microseconds) than did receptors occupied by EITC-oLH (43 microseconds). At this temperature both TRITC-oLH and TRITC-hCG occupied LH receptors were laterally immobile. These FPR and PFD results suggest that the molecular motions of the luteal cell LH receptor are significantly modulated by the subtle structural differences in various bound gonadotropins.

Class I major histocompatibility complex antigens are not associated with the LH/CG receptor on ovine luteal cells.

We have examined the rotational dynamics of the luteinizing hormone (LH) receptor on day 10 intact ovine small luteal cells and isolated plasma membranes using polarized fluorescence depletion (PFD). This technique measures rotational correlation times which are proportional to the in-membrane volume of a protein and are useful for examining changes in protein size due to receptor aggregation or protein-protein interactions. Eosin isothiocyanate (EITC)-derivatized ovine LH (EITC-oLH) bound to the LH receptor on luteal cell plasma membranes had a rotational correlation time of 20 +/- 6 microseconds, while that for EITC-human chorionic gonadotropin (EITC-hCG)-occupied LH receptors was 46 +/- 13 microseconds. Slower rotational times for EITC-oLH and EITC-hCG, 63 +/- 19 and 87 +/- 20 microseconds, respectively, were obtained on intact ovine luteal cells. These results indicate that the LH receptor exists as a larger molecular mass complex when binding hCG than oLH, a difference which could be attributable to hCG-induced LH-receptor interaction with additional membrane protein(s). One candidate protein for such an interaction is the Major Histocompatibility Complex (MHC) Class-I antigen. However, the rotational correlation time of EITC-anti-MHC Class-I antibody (SBU I) Fab fragments was 247 +/- 34 microseconds, indicating that MHC Class I is located in complexes larger than those identified by EITC-OLH or EITC-hCG. Preincubation of plasma membranes with 1 nM unlabeled oLH or hCG had no significant effect on this rotational correlation time. Further, treatment of cells with SBU I had no affect on either basal or oLH-stimulated progesterone secretion. Thus it appears that the ovine luteal LH-receptor is not associated with MHC Class I and that antibody-induced aggregation of MHC Class I does not cause an LH-mimetic response.

5-iodonaphthyl-1-azide labeling of plasma membrane proteins adjacent to specific sites via energy transfer.

We have examined conditions optimal for 5-iodonaphthyl-1-azide (INA4) labeling of membrane proteins proximal to known membrane sites. Membrane-bound INA can be indirectly activated by energy transfer from visible chromophores. We demonstrate that the efficiency of this sensitized activation is enhanced by use of triplet-forming chromophores such as eosin and by deoxygenation. Variation of sensitized activation efficiency with INA concentration indicates that the critical distance for eosin-INA energy transfer in solution is 8-14 A. We suggest that photosensitization occurs through triplet exchange and present an improved labeling protocol based on these findings. This protocol was used to examine whether different accessory proteins are associated with isolated and crosslinked Type I Fc epsilon receptors on 2H3 rat basophilic leukemia cells. 2H3 cells were incubated with eosin-conjugated IgE and irradiated at 514 nm yielding [125I]INA derivatized peptides at 53, 38, 34, and 29 kDa. Crosslinking IgE with mouse anti-rat IgE prior to irradiation labeled three additional proteins at 60, 54, and 43 kDa. These results demonstrate the utility of sensitized INA labeling in characterizing protein-protein interactions in membranes of intact cells and indicate the importance of considering photophysical factors when selecting sensitizers and reaction conditions. We discuss estimation of the size of the membrane region surrounding a sensitizing chromophore within which INA labeling of membrane proteins occurs.

Luteinizing hormone receptors are associated with non-receptor plasma membrane proteins on bovine luteal cell membranes.

Biophysical studies of the bovine luteinizing hormone (LH) receptor on luteal cell membranes suggest that this receptor may be part of a larger molecular weight structure. We have used 5-iodonaphthyl-1-azide (INA) to identify plasma membrane proteins near LH receptors on plasma membranes from bovine corpora lutea. Following binding of eosin isothiocyanate-derivatized ovine LH or human chorionic gonadotropin (hCG), five proteins with molecular weights of 71, 57, 55, 49 and 36 kDa were selectively derivatized with [125I]-INA following 2 h exposure at 22 degreesC to 514 nm light. However, there was no fluorescence energy transfer between LH receptors occupied by ovine LH or hCG indicating that LH receptors were not self-associated in these membrane preparations. Together these results suggest that, following hormone binding, single copies of the LH receptor may exist in large molecular weight structures that include non-receptor proteins.

Rotational dynamics of luteinizing hormone receptors and MHC class I antigens on murine Leydig cells.

We have examined the molecular motions of luteinizing hormone (LH) receptor and the Major Histocompatibility Complex Class I antigen on murine Leydig cells. Using time-resolved phosphorescence anisotropy methods, erythrosin (ErITC)-derivatized ovine luteinizing hormone (oLH) bound to the LH receptor appears rotationally mobile with rotational correlation times of 19.6 +/- 1.3 microseconds, 13.3 +/- 2.4 microseconds, 9.5 +/- 0.7 microseconds and 4.7 +/- 0.5 microseconds at 4 degrees C, 15 degrees C, 25 degrees C and 37 degrees C, respectively. Rotational correlation times for human chorionic gonadotropin (hCG)-occupied LH receptors were similar to those of the ErITC-oLH occupied receptor at each temperature. In addition, both oLH- and hCG-occupied LH receptors were laterally mobile in fluorescence photobleaching recovery experiments with diffusion coefficients at 29 degrees C of (5.8 +/- 0.9) x 10(-10) cm2 s-1 and (2.9 +/- 0.4) x 10(-10) cm2 s-1, respectively. We also measured the rotational correlation time of Class I antigen on murine Leydig cells using ErITC-derivatized 34-12-2S, an anti-Class I monoclonal antibody. Because there was no decay of the anisotropy function at 4 degrees C, 15 degrees C, 25 degrees C or 37 degrees C in the absence of oLH or following preincubation of Leydig cells with 1 nM oLH, it appears that Class I is rotationally immobile on the 1 ms timescale of our experiments. This result is consistent with the presence of Class I antigen in large molecular weight structures and may be the result of Class I self-aggregation. Further, treatment of cells with anti-Class I antibody had no effect on either basal or oLH-stimulated testosterone secretion. Thus, it appears that this anti-Class I antibody is not LH-mimetic on murine Leydig cells.

Expression and hormonal regulation of transcription factors GATA-4 and GATA-6 in the mouse ovary.

Two members of the GATA-binding family of transcription factors, GATA-4 and GATA-6, are expressed in the vertebrate ovary. To gain insight into the role of these factors in ovarian cell differentiation and function, we used in situ hybridization to determine the patterns of expression of GATA-4 and GATA-6 in mouse ovary during development and in response to hormonal stimulation. GATA-4 messenger RNA (mRNA) was first evident in the ovary around the time of birth. In the adult ovary, abundant GATA-4 mRNA was detected in granulosa cells of primary and antral follicles, with lesser amounts of GATA-4 message detected in theca cells, germinal epithelium, and interstitial cells. Little or no GATA-4 mRNA was found in corpus luteum. GATA-6 message exhibited a different distribution in the ovary, with abundant expression evident in both granulosa cells and corpora lutea. Stimulation of 3-week-old females with PMSG or estrogen enhanced follicular expression of GATA-4 and GATA-6 transcripts. Subsequent induction of ovulation with human CG resulted in a decrease in GATA-4 mRNA expression in granulosa cells, whereas GATA-6 mRNA expression persisted in granulosa cells after ovulation and in corpora lutea. Moreover, follicular apoptosis was associated with a decrease in the expression of GATA-4 but not GATA-6 message. Stimulation of cultured gonadal cell lines with FSH resulted in increased expression of GATA-4 message, whereas GATA-6 mRNA expression was not affected. In light of these findings, the established role of other GATA-binding proteins in hematopoetic cell differentiation and apoptosis, and the presence of conserved GATA motifs in the promoters of genes expressed selectively in ovary, we propose that GATA-4 and GATA-6 play distinct roles in follicular development and luteinization.