Cranio-cervical junction malformation causing cord compression in infant with achondroplasia: a bigger picture.

INTRODUCTION: Achondroplasia is a genetic disorder known for short stature and skeletal abnormalities. CASE REPORT: We present a case of narrowing of the foramen magnum from a large opisthion extending to the spinal canal. CONCLUSION: Foramen magnum stenosis and cervicomedullary stenosis are potentially life threatening neurological manifestations of achondroplasia.

Differential responses of one hundred tomato genotypes grown under cadmium stress.

Due to increased global concern over the deleterious effects of toxic heavy metals in the environment, it has become necessary to develop plant genotypes that limit the uptake of heavy metals to aerial edible parts. To address this concern, we performed a glasshouse experiment to assess variations within tomato germplasm for cadmium (Cd) tolerance under control conditions and under simulated stress conditions. Significant differences (P < 0.01) were observed among all genotypes at both Cd levels (3 ppm and 6 ppm). Our analyses showed that the genotypes 9086, Roma, Sitara TS-01, pak0010990, CLN-2123A, Picdeneato, 0.006231, and 7035 gave the best yields with minimum Cd content in their fruit, whereas the genotypes 42-07, 17883, BL-1176-Riostone-1-1, Marmande, and 17882 had relatively low yields with higher metal contents. The heavy metal was found to accumulate first in the shoot, then fruit, leaf, and finally root in tolerant genotypes; in susceptible genotypes, the order was fruit, shoot, leaf, and root. The inter-genotype differences in Cd uptake indicated the possibility of manipulating tomato genotypes to develop Cd tolerant tomato varieties or hybrids that allow safe use of a tomato crop grown on Cd contaminated soils.

Prevention of graft-tunnel mismatch during anatomical anterior cruciate ligament reconstruction using a bone-patellar tendon-bone graft.

Graft-tunnel mismatch of the bone-patellar tendon-bone (BPTB) graft is a major concern during anatomical anterior cruciate ligament (ACL) reconstruction if the femoral tunnel is positioned using a far medial portal technique, as the femoral tunnel tends to be shorter compared with that positioned using a transtibial portal technique. This study describes an accurate method of calculating the ideal length of bone plugs of a BPTB graft required to avoid graft-tunnel mismatch during anatomical ACL reconstruction using a far medial portal technique of femoral tunnel positioning. Based on data obtained intra-operatively from 60 anatomical ACL reconstruction procedures, we calculated the length of bone plugs required in the BPTB graft to avoid graft-tunnel mismatch. When this was prevented in all the 60 cases, we found that the mean length of femoral bone plug that remained in contact with the interference screw within the femoral tunnel was 14 mm (12 to 22) and the mean length of tibial bone plug that remained in contact with the interference screw within the tibial tunnel was 23 mm (18 to 28). These results were used to validate theoretical formulae developed to predict the required length of bone plugs in BPTB graft during anatomical ACL reconstruction using a far medial portal technique.

Diurnal expression of the thrombopoietin gene is regulated by CLOCK.

BACKGROUND: Most physiologic processes exhibit diurnal fluctuations controlled by the circadian regulation of sleep-wake behavior and feeding cycles. In addition, many cell types express endogenous circadian rhythms that affect cell-specific processes. Independent reports support the hypothesis that thrombopoietin (TPO) is under circadian control. OBJECTIVES: The current study tested the hypothesis that CLOCK, a circadian transcription factor, may regulate Thpo, the gene encoding TPO. METHODS: Circadian gene expression patterns were analyzed in mice and in human cell lines, Small interfering RNA was used to knock down CLOCK expression in cell lines, and gene expression was also examined in Clock(Δ19/Δ19) mutant mice. RESULTS: It was found that there was a diurnal rhythm in the expression of Thpoin vivo in mice, and that this was associated with concomitant rhythms of protein abundance. Thpo was rhythmically expressed in human cell lines, consistent with the gene being directly or indirectly regulated by the circadian clock. Silencing of CLOCK in the Huh7 human hepatoma cell line led to a significant reduction in the rhythmicity of Thpo expression. The expression of Mpl in murine marrow also displayed diurnal rhythmicity in vivo. In Clock(Δ19/Δ19) mutant mice, Thpo and Mpl expression was disrupted and there was an increase in the number of mature megakaryocytes, but no change in the ploidy distribution within the megakaryocyte population. CONCLUSIONS: These findings establish that Clock regulates Thpo and Mpl expression in vivo, and demonstrate an important link between the body's circadian timing mechanisms and megakaryopoiesis.

Evaluation of existing curriculum (2002) of undergraduate medical education in Bangladesh.

This study was conducted for evaluation of existing MBBS curriculum (2002) of undergraduate medical education in Bangladesh. The specific objectives of this study were: i) to assess the subject wise course content coverage in the new MBBS curriculum, ii) to assess different examination system for evaluation of MBBS students, iii) to evaluate the effectiveness of teaching and learning activities under the curriculum, iv) to explore students opinions regarding improvement of new curriculum. This was a descriptive cross-sectional study. The study was conducted among the students of Dhaka medical colleges of Bangladesh in 2008. Data was collected by self administered structured questioner adopting convenient sampling method. About ninety percent students opined that the coverage of course content of subjects in the curriculum in Phase I was enough. In case of the subjects in phase II except community medicine more than four fifth of the students expressed their opinion about coverage of course content in the curriculum as enough. In case of phase III it was mentioned by most of the students that coverage of course content was enough. Study revealed that teaching methods were perceived suitable by about three fourth of the respondents, to achieve learning objectives. Most of the students expressed their positive views regarding practice of block posting teaching. More than three fourth of the students perceived that formative assessment was encouraging for students to become time bound learner and Structured Oral Examination (SOE) was fair on an average. Only 31(8.6%) of the respondents had opinion that Objective Structured Practical Examination (OSPE)/Objective Structured Clinical Examination (OSCE) was not well organized. About half of the students opined that 20% marks in written test should be allocated for Multiple Choice Question (MCQ). Students' suggestions regarding teaching were: there should be smaller group sessions; more interactive sessions; more clinical and practical sessions; more problem oriented sessions; more sessions with senior and experienced teachers; teachers should follow the curriculum properly; and should be well prepared for class. Regarding assessment suggestions were: written script of the formative examination should be returned to students with feedback; teachers should not be biased. Study recommended that training of the teachers on teaching methodology and assessment system is needed; teachers should provide feedback to the students according to the performance of the formative assessment at the individual level; to maintain the standards of assessment proper planning, designing, conduction and evaluation of assessment should be taken into consideration; subject wise review and updating is essential to make the curriculum more need based, user friendly and applicable considering context of Bangladesh.

A 30 year old female with lower abdominal lump.

A female of 30 years of age, hailing from Narayangonj, Dhaka got admitted herself into Dhaka medical College & Hospital on 25.10.08 with the complaints of a lump in the lower abdomen for 1 year and pain in the lower abdomen for the same duration. Local examination reveals a lump in the lower abdomen which is intra abdominal, 10cm × 12cm in size. CT scan of Abdomen reveals a solid mass measuring (12 × 9 × 8.5) cm is seen in left side of abdominal cavity extending up to pelvic cavity. FNAC of abdominal lump shows sheets of mesothelial cells and few small clusters of regular epithelial like cells. Laparotomy was done on 27.11.08 under G/A & reveals a solid mass measuring about 10 × 8cm size & was well encapsulated, adherent with omentum found in the lower abdomen. Few mesenteric lymph nodes were enlarged. The mass was gently separated from the omentum ensuring adequate haemostasis. Post-operative period was uneventful. Histopathology report shows neoplasm composed of plump of fibroblasts arranged in broad sweeping fascicles and infiltrate into adjacent tissue consistent with fibromatosis. The patient was discharged with advice on 10th post operative day.

Genetic diversity in merozoite surface protein-1 and 2 among Plasmodium falciparum isolates from malarious districts of tribal dominant state of Jharkhand, India.

INTRODUCTION: The genetic make-up of malaria parasite is potent for understanding the parasite virulence, designing antimalarial vaccine and evaluating the impact of malaria control measures. There is a paucity of information on genetic structure of Plasmodium falciparum in Jharkhand, India where malaria is rampant and this study aimed to establish molecular characterization of P. falciparum field isolates from Jharkhand measured with two highly polymorphic genetic markers, i.e. the merozoite surface proteins (MSPs) 1 and 2. METHODS: The genetic diversity of P. falciparum population from low transmission area, Ranchi, Bokaro and Hazaribagh and highly malarious area, Latehar and Palamau districts of Jharkhand were evaluated by polymerase chain reaction-sequencing analyzing msp-1 and msp-2 genes to explore the genetic structure of parasite from this understudied region. RESULTS: A total of 134 P. falciparum isolates were analyzed by polymorphic regions of msp-1 and msp-2 and classified according to prevalence of allelic families. The majority of patients from all the five sites had mean monoclonal infections of 67·1 and 60·4% of P. falciparum for msp-1 and msp-2, respectively, whereas, mean multiple genotypes of 32·8 and 39·5% for msp-1 and msp-2, respectively. Interestingly, we observed higher multiclonal infection in low transmission area as compared to highly malarious area in the case of msp-1 genotypes, whereas in msp-2 higher multiclonal infection was observed in highly malarious area compared to low transmission area. The overall multiplicities of infection of msp-1 and msp-2 were 1·38 and 1·39, respectively. CONCLUSION: This is the first report on molecular characterization of P. falciparum field isolates from Jharkhand. The genetic diversity and allelic distribution found in this study is somewhat similar to other reports from India and Southeast Asian countries. However, P. falciparum infection can be highly complex and diverse in these disease-endemic regions of Jharkhand, suggesting continual genetic mixing that could have significant implications for the use of antimalarial drugs and vaccines.

A 40 year old man with acute abdomen.

A 40 years old man with the complaints of abdominal pain and per rectal bleeding for 2 days and abdominal distension for 1 day. There was an ill defined, tender intra abdominal solid mass at the left side of the lower abdomen measuring about 3 x 3cm2 on deep palpation. Direct rectal examination could not be done due to excessive pain at the anal region and non co operation of the patient. Plain X-ray of the abdomen A/P view in erect posture including both domes of the diaphragm shows distended large intestinal gas shadow & bottle like radiopaque shadow at the lower mid abdomen. Ultrasonogram (USG) of the abdomen could not be done due to the restlessness of the patient. With all available aseptic precaution the abdomen was opened by a lower midline incision. After exploration of the sigmoid colon two glass bottles (One measuring 10cm x 5 cm, the other 12cm x 7cm) were removed and the colon was closed primarily. There was also a small piece of stone found in the peritoneal cavity measuring 2 cm in diameter which was sent for histopathology. Post operative period was uneventful. The patient was discharged with advice on the 8th POD.

Treatment modalities of TMJ ankylosis: experience in Delta Nile, Egypt.

The study reports the authors' experience in managing TMJ ankylosis in Delta Nile, Egypt (1995-2006) and compares the surgical modalities used. 101 patients (109 joints) were reviewed in this retrospective study. Pre- and postoperative assessment included history, radiological and physical examination, and mouth opening. Age, sex, aetiology, joint(s) affected, surgical modality, complications and follow up periods were evaluated. Various types (fibrous, fibro-osseous and bony) of TMJ ankylosis were diagnosed; trauma was the commonest aetiology. The patients' age range was 2-41 years, 62% were female, and the follow up period ranged from 14 to 96 months. Average mouth opening was significantly increased from 5.3mm pre-operatively to 32.9 mm 12 months postoperatively (P=0.0001). Marked improvement in mouth opening was documented when the ramus-joint complex was reconstructed using distraction osteogenesis (34.7 mm), costochondral graft (34.4mm) and Surgibone (34.6mm). Gap arthroplasty showed least satisfactory mouth opening compared with other techniques (P=0.001). Minor and major complications were encountered in 33% of cases, including 5% recurrence rate. Early release of TMJ ankylosis; reconstruction of the ramus height with distraction osteogenesis or bone grafting combined with interpositional arthroplasty, followed by vigorous physiotherapy is successful for managing TMJ ankylosis.

Management of external hernias: analysis of 1020 [corrected] cases.

A prospective study was done to find out the incidence of different external hernias, identify the predisposing factors and type of repair performed. Over a period of 8 years, 1020 patients were admitted with different varieties of external hernias in Chittagong Medical College Hospital. Among them 743 patients had inguinal hernia, 130 had incisional hernia, 58% had indirect inguinal hernia, 29% had direct inguinal hernia, Bilateral inguinal hernia was present in 13% cases. 272 (32.08%) presented with complications like, irreducibility, obstruction, strangulation, 576 (67.92%) was admitted from out-patient department for elective operation. 97.04% of patients with inguinal hernia were male, 2.96% of patients were female. But among incisional hernia 77.70% are female and 22.30% are male patients. The highest incidence of both inguinal hernia and incisional hernia were found in 4th decade of life. Predisposing factors could be identified in 42.06% cases. 90.76% patients approached doctors for advice after 6 months of noticing hernia only 36.37% could mention the cause of delay. Out of 1020 patients (812) 72.18% patients underwent different operations. 2.5% patients were inoperable due to associated disease like Ca-liver, IHD, renal transplant, CLD, uncontrolled DM. Others refused or absconded, being afraid of operation. Effort for post operative follow up was not very satisfactory as only a small number could be followed for a limited period.

Hypoxia and pulmonary acclimatisation at 4578 m altitude: the role of acetazolamide and dexamethasone.

OBJECTIVES: To quantify the changes in ventilatory response and arterial blood gases in healthy male volunteers on acute ascent to 4578 meters altitude along with evaluating the role of acetazolamide and dexamethasone prophylaxis in acclimatisation. METHODS: Forty four lowlander male subjects participated as two groups in the study. Twenty four lowlander healthy male adults (age mean +/- SE 27.8 +/- 1.24 years) comprised the non-acclimatised group. They were sub grouped in a double blind fashion into four. Each subgroup (n=6) received placebo (multivitamin) or acetazolamide (250 mg) or dexamethasone (4 mg) or combined regimen of the two drugs twice daily for 5 days commencing 24 hours before ascent. The volunteers reached the altitude of 4578 meters within a span of one day. The second group (acclimatised) comprised of age and height matched twenty volunteers who had arrived 4 and 8 weeks earlier at the same altitude. Arterial PO2, PCO2, SO2 and minute respiratory rate pH were measured. The pulmonary functions (FVC, %FEV1 PEF, FEF(25-75%) and MVV) were recorded by Compact Spirometer (Vitalograph). Pre and post ascent measurements were carried out in non-acclimatised group whereas one measurement of the same parameters were made in acclimatised group. RESULTS: The study revealed a significant increase in respiratory rate min-1 after 24 hours of ascent along with reduction in PaO2 and SaO2. The gradual increase in PaO2 and SaO2 and reduction in PaCO2 was observed after 4 weeks at the same altitude. The reduction in FVC, %FEV1, PEF, FEF(25-75%) and MW were recorded in our subjects after 24 hours of acute ascent. Nevertheless, all pulmonary parameters in volunteers having 8 weeks of stay at 4578 m altitude, were found equivalent to the non-acclimatised group. The greater increase in minute respiratory rate, PaO2, SaO2 and pulmonary functions was found in volunteers taking acetazolamide. CONCLUSION: It is concluded that hyperventilation and increase in lung volumes are the adaptive pulmonary responses which help in improving levels of PaO2 and SaO2. The acetazolamide-dexamethasone prophylaxis appears beneficial in promoting pulmonary acclimatisation during hypobaric hypoxia.

The disposition of diclofenac in camels after intravenous administration.

The pharmacokinetics of diclofenac was studied in camels (Camelus dromedarus) (n=6) following intravenous (i.v.) administration of a dose of 2.5 mg kg(-1) body weight. The metabolism and urinary detection time were also studied. The results obtained (median and range) were as follows: the terminal elimination half-life (t(1/2beta)) was 2.35 (1.90-2.73)h, total body clearance (Cl(T)) was 0.17 (0.16-0.21)lh kg(-1). The volume of distribution at steady state (V(SS)) was 0.31 (0.21-0.39)l(-1)kg(-1), the volume of the central compartment of the two compartment pharmacokinetic model (V(C)) was 0.15 (0.11-0.17)l kg(-1). Five metabolites of diclofenac were tentatively identified in urine and were excreted mainly in conjugate form. The main metabolite was identified as hydroxy diclofenac. Both diclofenac and hydroxy diclofenac, appear to be the main elimination route for diclofenac when administered i.v. in camels. Diclofenac could be identified up to 4 days following i.v. administration in camels using a sensitive gas chromatography/mass spectrometry (GC/MS) method.

Comparative pharmacokinetics of diphenhydramine in camels and horses after intravenous administration.

The pharmacokinetics of diphenhydramine (DPHM) was compared in camels (n = 8) and horses (n = 6) following intravenous (i.v.) administration of a dose of 0.625 mg/kg body weight. In addition, the metabolism and urinary detection time of DPHM was evaluated in camels. The data obtained (median and range in brackets) in camels and horses, respectively, were as follows. The terminal elimination half lives (h) were 1.58 (1.13-2.58) and 6.11 (4.80-14.1), and the total body clearances (L/h per kg) were 1.42 (1.13-1.74) and 0.79 (0.66-0.90). The volumes of distribution at steady state (L/kg) were 2.38 (1.58-4.43) and 5.98 (4.60-8.31) and the volumes of the central compartment of the two compartment pharmacokinetic model were 1.58 (0.80-2.54) and 2.48 (1.79-3.17). All the pharmacokinetic parameters in camels were significantly different from those of horses. Five metabolites of DPHM were tentatively identified in the camel's urine. Two metabolites, diphenylmethoxyacetic acid and 1-(4-hydroxyphenyl)-phenylmethoxyacetic acid, were present in the acid fraction. Two metabolites, desamino-DPHM and diphenylmethanol, were identified in the basic fraction, in addition to DPHM itself, which was present mainly as a conjugate. Even after enzymatic hydrolysis, DPHM could be detected for up to 24 h in camels after an i.v. dose of 0.625 mg/kg body weight.

Prevalence, risk factors and genotype distribution of HCV and HBV infection in the tribal population: a community based study in south India.

Viral hepatitis caused by the hepatitis C virus (HCV) and hepatitis B virus (HBV) represents a major public health problem in India. These viruses share common modes of transmission, such as parenteral routes. We aimed to assess the exposure of a tribal population to these viruses in south India. The present study was carried out on serum samples from 890 individuals (526 males and 324 females) belonging to the Lambada tribe residing in the state of Andhra Pradesh, south India. Anti-HCV antibody and hepatitis B surface antigen (HBsAg) status in the sera were analyzed using commercially available enzyme immunoassays (Abbott Labs, Chicago, IL). HCV-RNA and HBV-DNA in the sera was tested by reverse transcriptase polymerase chain reaction (RT-PCR) and PCR, respectively. The infecting genotype of HCV was determined using type-specific primers corresponding to the NS5 region of the virus. Out of the 890 samples, 18 (2.02%; male 11/526; female 7/364) were positive for HCV-RNA by RT-PCR and, 17 of them were positive for anti-HCV antibody. Genotyping of HCV isolates from the 18 individuals positive for HCV-RNA revealed that 66.67% (12/18) were infected with type 1 of HCV and its variants; while in the remaining (6/18), the infecting genotype was found to be type 3 and its variants. A total of 46 samples (5.16%; males 28/526; female 18/364) were positive for HBsAg; while 11 were positive only for HBV-DNA, 9 were positive for both hepatitis B e antigen (HBeAg) and HBV-DNA. Cultural practices such as tattooing, traditional medicine (e.g. blood-letting), rituals (e.g. scarification), body-piercing etc are the potential sources of spread of infection in this tribe. None of the samples analyzed revealed co-infection with the 2 viruses.

Acute mountain sickness score and hypoxemia.

BACKGROUND: Hypoxemia is the immediate consequence of hyobaric hypoxia, which is the crucial starting mechanism of acute mountain sickness (AMS). The AMS is generally a benign and self-limiting condition which can be prevented by gradual ascent. However, ascent rates recommended for prophylaxis of AMS are far slower than those attempted during military operations and by climbers. OBJECTIVE: The current study was carried out to quantify the relationship between AMS and hypoxemia alongwith evaluating the benefits of acetazolamide-dexamethasone chemoprophylaxis during acute ascent. SUBJECTS AND METHODS: Twenty-four low lander male adults (age mean +/- SE 27.8 +/- 1.24 years) were selected. They were grouped in a double-blind fashion into four groups and each group (n = 6) received placebo (multivitamin) or acetazolamide (250 mg) or dexamethasone (4 mg) or a combined regimen of the two drugs twice daily for 5 days, commencing 24 hours before ascent. The volunteers reached the altitude of 4578 meters within a span of one day. Their AMS symptoms were recorded on modified environmental symptoms questionnaire (ESQ), after 24 and 72 hours of ascent. Arterial PO2, SO2 and PCO2 were measured by GEMSTAT blood-gas analyzer (Mallincrodt-USA). RESULTS: The ESQ, AMS-C (cerebral) and AMS-R (respiratory) scores of combined therapy group were significantly lower as compared to the other groups on the symptom rating scale. The significant finding amongst the volunteers taking acetazolamide was mild to moderate diuresis whereas severity of headache was markedly less in dexamethasone group. The commonest feature of combined therapy was that none of the volunteers complained of headache, dysponea, irritability and more than mild disturbance of sleep. The ESQ scores of volunteers were inversely correlated to PaO2 and SaO2 after 24 hours of ascent to 4578 meters. CONCLUSION: The study concludes that severity of AMS is closely related to hypoxemia and combination therapy of acetazolamide-dexamethasone may be effective in preventing AMS.

Binding of microsomal triglyceride transfer protein to lipids results in increased affinity for apolipoprotein B: evidence for stable microsomal MTP-lipid complexes.

Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are known to interact with each other. We evaluated the effect of different lipids on the protein-protein interactions between MTP and apoB100 or its C-terminally truncated forms. Negatively charged lipids decreased protein-protein interactions between apoB and MTP. In contrast, zwitterionic phospholipids enhanced (2-4-fold) the binding of apoB100 to MTP by increasing affinity (1.5-3-fold) between these proteins without affecting the number of binding sites. Similarly, phospholipids augmented (1.5-4-fold) the binding of various C-terminally truncated apoB peptides to MTP. The increased binding was greater for apoB peptides containing lipid-binding domains, such as apoB28 and apoB42. Surprisingly, preincubation of apoB28 with lipid vesicles had no effect on MTP binding. In contrast, incubation of MTP with lipid vesicles resulted in a stable association of MTP with vesicles, and MTP-lipid vesicles bound better (5-fold increase) to LDL than did lipid-free MTP. To determine whether MTP exists stably associated with lipids in cells, microsomal contents from COS cells expressing MTP, HepG2 cells, and mouse liver were ultracentrifuged, and MTP was visualized in different density fractions. MTP was found associated and unassociated with lipids. In contrast, apoB17 and apoB:270-570 were present unassociated with lipids in COS cells. These studies show that the binding of MTP to lipids results in increased affinity for apoB and that stable MTP-lipid complexes exist in the lumen of the endoplasmic reticulum. Protein-protein interactions between apoB and MTP may juxtapose lipids associated with MTP to lipid-binding domains of apoB and facilitate hydrophobic interactions leading to enhance affinity. We speculate that MTP-lipid complexes may serve as nuclei to form "primordial lipoproteins" and may also play a role in the bulk addition of lipids during the "core expansion" of these lipoproteins.

Mechanisms involved in the intestinal digestion and absorption of dietary vitamin A.

Dietary retinyl esters are hydrolyzed in the intestine by the pancreatic enzyme, pancreatic triglyceride lipase (PTL), and intestinal brush border enzyme, phospholipase B. Recent work on the carboxylester lipase (CEL) knockout mouse suggests that CEL may not be involved in dietary retinyl ester digestion. The possible roles of the pancreatic lipase-related proteins (PLRP) 1 and 2 and other enzymes require further investigation. Unesterified retinol is taken up by the enterocytes, perhaps involving both diffusion and protein-mediated facilitated transport. Once in the cell, retinol is complexed with cellular retinol-binding protein type 2 (CRBP2) and the complex serves as a substrate for reesterification of the retinol by the enzyme lecithin:retinol acyltransferase (LRAT). Retinol not bound to CRBP2 is esterified by acyl-CoA acyltransferase (ARAT). The retinyl esters are incorporated into chylomicrons, intestinal lipoproteins that transport other dietary lipids such as triglycerides, phospholipids, and cholesterol. Chylomicrons containing newly absorbed retinyl esters are then secreted into the lymph.

Metabolism of equilenin in MCF-7 and MDA-MB-231 human breast cancer cells.

Sulfate conjugates of the B-ring unsaturated estrogens, equilin, equilenin, and 8-dehydroestrone, and their 17alpha- and 17beta-dihydro analogues, constitute about 54% of Premarin (Wyeth-Ayerst), the most commonly prescribed estrogen formulation in estrogen replacement therapy. Despite the wide clinical use of Premarin, there have been very few studies on the metabolism of the B-ring unsaturated estrogens in humans and there is no information regarding the fate of these compounds in breast tissue or tumors. In this study, we investigated the metabolism of equilenin in two lines of human breast-cancer cells, MCF-7 and MDA-MB-231. MCF-7 cells respond to treatment with Ah-receptor agonists with induction of cytochromes P450 1A1 and 1B1, whereas in MDA-MB-231 cells P450 1B1 is predominantly induced. Metabolites of equilenin were identified and quantified by GC/MS utilizing a series of synthetic metabolite standards and deuterium-labeled analogues as internal standards. In the two cell lines, the same pathways of equilenin metabolism were observed. Equilenin was reduced at C-17 to the 17beta-dihydro form, with minimal production of the 17alpha-dihydro isomer. Both equilenin and 17beta-dihydroequilenin were hydroxylated at the C-4 position, and the resultant catechol metabolites were methylated to form 4-methoxyequilenin and 4-methoxy-17beta-dihydroequilenin. Rates of equilenin metabolism were markedly elevated in cultures exposed to the Ah-receptor agonists, 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3,4,4',5-tetrachlorobiphenyl, implicating the activities of P450s 1A1 and 1B1 in the metabolism. The 2-hydroxylation pathways of equilenin and 17beta-dihydroequilenin metabolism were not observed. In microsomal reactions with cDNA-expressed human enzymes, both P450s 1A1 and 1B1 catalyzed the 4-hydroxylation of 17beta-dihydroequilenin, whereas with 17beta-estradiol as substrate P450 1A1 catalyzes predominantly 2-hydroxylation and P450 1B1 predominantly 4-hydroxylation. Since P450 1B1 is constitutively expressed and both P450s 1A1 and 1B1 are inducible in many extrahepatic tissues including the mammary epithelium, these results indicate the potential for 4-hydroxylation of equilenin and 17beta-dihydroequilenin in extrahepatic, estrogen-responsive tissues.

Structural, biochemical and signaling properties of the low-density lipoprotein receptor gene family.

The low-density lipoprotein (LDL) receptor (LDL-R) family members (LDL-R, LRP, megalin, VLDL-R, apoER2) bind several extra-cellular structurally dissimilar ligands and internalize them for degradation by lysosomes by a process called receptor-mediated endocytosis. The receptor-mediated endocytosis involves immobilization of circulating ligands onto the cell-surface followed by their internalization and degradation. All the receptors can perform both of these functions. However, in the majority of the cases, other proteins immobilize ligands on to the cell-surface and subsequent internalization is mediated by these receptors. The LDL-R and LRP play important roles in plasma cholesterol homeostasis and fetal development. Megalin is an antigenic determinant for Heymann nephritis in rats and may be important for re-absorption of various molecules by the kidney. VLDL-R homologue in chicken is essential for female fertility. This receptor and apoER2 are critical for the proper development of the brain in mice. The members of the LDL-R gene family contain several complement-type and EGF precursor-like repeats, and single transmembrane and cytoplasmic domain. Cysteine-rich complement-type repeats containing DxSDE sequences at the C-termini constitute ligand-binding domains. In contrast to the ligand binding domains, receptor-binding domains in different ligands do not share sequence homology. It has been proposed that positive electrostatic surface potentials, not the primary sequences, in different ligands constitute receptor-binding domains. The EGF precursor homology repeats in receptors are important for the dissociation of ligands from receptors in endocytic vesicles. The transmembrane domain is necessary for anchoring to membranes and the cytoplasmic domain is required for their targeting to coated pits and subsequent internalization. The receptor-mediated endocytosis involves recognition of the NPXY motif by clathrin. Recently, this motif has also been implicated in signaling pathways that are crucial in brain development. The signaling process involves the recognition of the NPXY motif by Disabled-1 protein and possibly other proteins involved in intracellular signaling cascade. The LDL-R gene family has provided important insights into the mechanisms of ligand catabolism and may serve as new targets for the treatment of different cardiovascular and neuronal disorders. In the future, their role in signaling may provide novel insights into brain development and neuronal layering.

Signposts in the assembly of chylomicrons.

Intestinal cells synthesize and secrete chylomicrons in the postprandial state. Synthesis of these particles is defective in abetalipoproteinemia and chylomicron retention disease. Chylomicrons are very large, heterogeneous, lipid-rich particles ranging in diameters from 75 to 450 nm and function to transport dietary fat and fat-soluble vitamins to blood. The size heterogeneity of the secreted particles depends on the rate of fat absorption, type and amount of fat absorbed. The fatty acid composition of triglycerides present in chylomicrons reflects the composition of dietary fat, whereas the fatty acid composition of chylomicron phospholipids does not. The differences in the fatty acid compositions are also observed when lipids are labeled with glycerol. Thus, the differences are not due to differential incorporation of dietary fatty acids into different lipids but are mainly due to different pools of lipids used for chylomicron assembly. It has been suggested that preformed phospholipids and nascent triglycerides are preferentially used for intestinal lipoprotein assembly. Biosynthesis of chylomicrons requires apoB48. ApoB48 is translated from apoB mRNA that is post-transcriptionally edited in the intestinal cells to incorporate a stop codon. Nascent apoB48 may be cotranslationally lipidated and this process is critically dependent on the presence of microsomal triglyceride transfer protein. Two different models have been proposed for the assembly of chylomicrons. In the independent model, intestinal cells are hypothesized to synthesize VLDL and chylomicron by two independent pathways. The chylomicron assembly pathway is hypothesized to be sensitive to a surfactant, Pluronic L81, but that of VLDL assembly is not. In the sequential assembly model, synthesis of all lipoproteins is hypothesized to begin with the assembly of apoB-containing primordial lipoprotein particles. The primordial particles are suggested to fuse with triglyceride-rich lipid droplets that are synthesized independently of apoB. This process results in the core expansion of primordial particles and the synthesis of nascent lipoproteins. Differences in the size of secreted lipoproteins may be due to differences in the size of triglyceride-rich lipid droplets. Pluronic L81 is hypothesized to inhibit the formation of large triglyceride-rich droplets that serve as precursors for chylomicron assembly. In this review, we have discussed some signposts that might be unique to different steps in the assembly of chylomicrons. First, it is proposed that the association of preformed phospholipids with nascent apoB in the endoplasmic reticulum may serve as a signpost for the very early steps in the assembly of chylomicrons. Second, association of large amounts of newly synthesized triglycerides compared to preformed triglycerides may serve as a signpost for the assembly of larger lipoproteins. Third, the incorporation of retinyl esters may serve as markers for the final stages of chylomicron assembly. These signposts may be helpful in the identification and characterization of various intermediates in the assembly of chylomicrons. The knowledge about the molecular assembly of chylomicrons may lead to better therapeutic agents for controlling various hyperlipidemias, obesity, and atherosclerosis.