Testosterone administration to adult rats differentially modulates androgen and oestrogen receptor-α expression in reproductive organs and pituitary.

Regulation of androgen receptor (AR) and oestrogen receptor α (ERα) expression has direct bearing on the physiology of male reproductive organs. With the help of three independent tools of immunohistochemistry, western blotting and RT-PCR, AR and ER α receptor expression was examined in the testis, epididymis, prostate, seminal vesicle and pituitary of adult rats following testosterone enanthate (TE, 3 mg/100 µl of olive oil/rat per week) intervention for 15 and 30 days. TE administration reduced AR immunoexpression which coincided well with the decline in the receptor protein and transcript levels. In contrast, ERα was found overexpressed in all the organs. While weights of testis and epididymis decreased significantly, those of prostate, seminal vesicle and pituitary demonstrated an upward trend. Spermatogenesis was adversely affected with decline in number of germ cells per tubule and increased prevalence of germ cell apoptosis. Increase in serum and decrease in intra-testicular levels of testosterone were found significant (P < 0.001) in both 15 and 30 days treatment groups. Serum follicle stimulating hormone declined significantly (P < 0.001) at the end of 30 days treatment. Taken together, the above findings indicate that the testosterone intervention differentially modulates, AR ERα expression, which is associated with hypospermatogenesis and increased germ cell apoptosis.

1alpha,25-dihydroxyvitamin D(3)-induced myeloid cell differentiation is regulated by a vitamin D receptor-phosphatidylinositol 3-kinase signaling complex.

1alpha,25-dihydroxyvitamin D(3) (D(3)) promotes the maturation of myeloid cells and surface expressions of CD14 and CD11b, markers of cell differentiation in response to D(3). To examine how these responses are regulated, THP-1 cells were grown in serum-free medium and incubated with D(3). This was associated with rapid and transient increases in phosphatidylinositol 3-kinase (PI 3-kinase) activity. Furthermore, induction of CD14 expression in response to D(3) was abrogated by (a) the PI 3-kinase inhibitors LY294002 and wortmannin; (b) antisense oligonucleotides to mRNA for the p110 catalytic subunit of PI 3-kinase; and (c) a dominant negative mutant of PI 3-kinase. In THP-1 cells, induction of CD11b expression by D(3) was also abrogated by LY294002 and wortmannin. Similarly, LY294002 and wortmannin inhibited D(3)-induced expression of both CD14 and CD11b in peripheral blood monocytes. In contrast to CD14 and CD11b, hormone-induced expression of the Cdk inhibitor p21 in THP-1 cells was unaffected by either wortmannin or LY294002. These findings suggest that PI 3-kinase selectively regulates D(3)-induced monocyte differentiation, independent of any effects on p21.

Effect of chronic oestrogen administration on androgen receptor expression in reproductive organs and pituitary of adult male rat.

Following chronic (15 or 30 days) treatment with oestradiol 3-benzoate (75 microg rat(-1) day(-1) in 100 microl of olive oil) to adult rats, androgen receptor (AR) expression was analysed simultaneously in testis, epididymis, seminal vesicle, prostate and pituitary utilising three independent tools i.e. immunohistochemistry, Western blotting and RT-PCR. All the five organs showed higher AR transcriptional activity gradually increasing from 15 to 30 days of oestrogen treatment. However, the AR protein expression either through immunostaining or Western blotting demonstrated a significant decline in all the reproductive organs. In the pituitary, on the other hand, the decline coincided with a distinct breakdown of the AR protein into two bands with increasing duration of treatment. Serum and intra-testicular testosterone levels were found significantly lowered. Spermatogenesis was adversely affected with concurrent decrease in weights of testis and accessory sex organs. Decrease in testis weight was consistent with the reduction in the number of maturing germ cells per tubule. Despite the decrease in weight, accessory sex organs like epididymis, seminal vesicle and prostate were completely devoid of any apoptotic cells which were characterised only in testis and pituitary. Seminiferous epithelium demonstrated a marked increase in the number of germ cells undergoing apoptosis. However, the rate of cell apoptosis was much higher in the pituitary than in the testis at the end of 30 days treatment. It is therefore concluded that degradation of AR protein expression after oestrogen treatment is probably directly linked to an increase in cell apoptosis both in testis and pituitary.

Ethyl-3,4-dihydroxybenzoate inhibits myoblast differentiation: evidence for an essential role of collagen.

To study the role of (pro)collagen synthesis in the differentiation of rat L6 skeletal myoblasts, a specific inhibitor of collagen synthesis, ethyl-3,4-dihydroxybenzoate (DHB), was utilized. It is shown that DHB reversibly inhibits both morphological and biochemical differentiation of myoblasts, if it is added to the culture medium before the cell alignment stage. The inhibition is alleviated partially by ascorbate, which along with alpha-ketoglutarate serves as cofactor for the enzyme, prolyl hydroxylase. DHB drastically decreases the secretion of procollagen despite an increase in the levels of the mRNA for pro alpha 1(I) and pro alpha 2(I) chains. Probably, the procollagen chains produced in the presence of DHB, being underhydroxylated, are unable to fold into triple helices and are consequently degraded in situ. Along with the inhibition of procollagen synthesis, DHB also decreases markedly the production of a collagen-binding glycoprotein (gp46) present in the ER. The results suggest that procollagen production and/or processing is needed as an early event in the differentiation pathway of myoblasts.

A sensitive human bone assay for quantitation of tigecycline using LC/MS/MS.

Tigecycline (Tygacil,Wyeth) is a first-in-class, broad spectrum antibiotic with activity against multiple-resistant organisms. In order to address the unexpectedly low tigecycline concentrations in human bone samples analyzed using a LC/MS/MS method developed elsewhere, we have developed and validated a new and sensitive human bone assay for the quantitation of tigecycline using LC/MS/MS. The new method utilizes the addition of a stabilizing agent to the human bone sample, homogenization of human bone in a strong acidic-methanol extraction solvent, centrifugation of the bone suspension, separation by liquid chromatography, and detection of tigecycline by mass spectrometry. Linearity was demonstrated over the concentration range from 50 to 20,000 ng/g using a 0.1g human bone sample. The intra- and inter-day accuracy of the assay was within 100+/-15%, and the corresponding precision (CV) was <15%. The stability of tigecycline was evaluated and shown to be acceptable under the assay conditions. The extraction recovery of tigecycline with the current method was 79% when using radio-labeled rat bone samples as a substitute for human bone samples. Twenty-four human bone samples collected previously from volunteers without infections who had elective orthopedic surgery after receiving a single dose of tigecycline were re-analyzed using the current validated method. Tigecycline concentrations in these samples ranged from 238 to 794 ng/g with a mean value 9 times higher than the mean concentration previously reported. The data demonstrated that the current method has significantly higher extraction efficiency than the previously reported method.

Release of copper from CuT 380A co-incubated with human semen and its effect on sperm function in vitro.

Release of copper and its effect on functional integrity of human sperms in vitro were assessed following co-incubation of semen with CuT 380A. After 30 min of incubation with semen, release of copper ions from CuT 380A was found to be 9.2 to 40 times higher compared to control incubations with PBS. Sperm function tests, when simultaneously performed following loss of motility in sperms (> 95%) after 120 min of copper exposure, depicted a significant (P < 0.001) reduction in sperm viability and hypo-osmotic swelling (HOS) response. However, the affected sperm populations revealed no significant alterations in other functional tests like acrosomal status or nuclear chromatin decondensation. It is therefore concluded that the high release of copper from CuT 380A drastically lowers sperm motility, viability and HOS response but only marginally affects the acrosome status or nuclear chromatin condensation in short term incubations.

A novel antibiotic bone assay by liquid chromatography/tandem mass spectrometry for quantitation of tigecycline in rat bone.

Tigecycline (Tygacil) is a first-in-class, broad spectrum antibiotic with activity against antibiotic-resistant organisms. In rats and humans, tigecycline readily distributes to bone tissue but its accuracy of quantitation via liquid chromatography/mass spectrometry (LC/MS/MS) is hindered by a low extraction recovery when using a conventional plasma extraction method. To overcome this issue, we have identified an effective extraction solvent for quantitation of tigecycline in rat bone. The current LC/MS/MS bone assay is novel, simple, and sensitive, and has a wide linear range of 50-10,000 ng/g. The assay requires homogenization of the rat bone in a strong acidic-methanol extraction solvent, centrifugation of the bone suspension, separation of the supernatant with liquid chromatography, and detection of tigecycline with tandem mass spectrometry. The incurred pooled rat bone samples obtained from rats given 3mg/kg/day of [(14)C]-tigecycline and non-radio-labeled tigecycline were analyzed with the current method. The absolute extraction recovery of the bone assay for tigecycline was 77.1%. The intra-day accuracy ranged from 91.7 to 106% with precision (CV) of 1.9-10.7%, and inter-day accuracy ranged from 96.1 to 100% with a precision of 6.3-8.7%. In addition, biological activity was demonstrated for the tigecycline extracted from incurred rat bone. This bone assay provides an important analytical tool for the determination of drug concentrations (especially, antimicrobials) in rodent bone tissues and has served as the foundation of development and validation of a similar bone assay for tigecycline in human bone tissues.

Complementary feeding practices in rural area of district Agra.

The study was conducted to assess the prevailing complementary feeding practices. Strikingly 30.9 percent infants were not receiving complementary foods. 21.9 percent mothers initiated complementary feeding at the right age. This was significantly associated with the literacy status of the mothers (p<0.05). 87.1 percent infants were receiving family pot feeding. The complementary foods fed to the infants were mostly (88.6%) of semi-solid consistency. Demand feeding was being practiced by 66.7 percent mothers. The practice of cleaning hands before feeding was very poor with only 17.1%. Thus to conclude the complementary feeding practices were not satisfactory in the rural area of Agra and need to be improved.

Exploitation of host cell signaling machinery: activation of macrophage phosphotyrosine phosphatases as a novel mechanism of molecular microbial pathogenesis.

Intracellular pathogens, particularly those that target host mononuclear phagocytes, have evolved strategies to either evade or inhibit cellular mechanisms of host defense. Mycobacterium tuberculosis and Leishmania donovani exemplify a diverse group of microorganisms that have developed the ability to invade and replicate within host macrophages, leading to disease expression. Recent studies have suggested that the pathogenesis of intracellular infection may involve interference with host cell signaling. Drawing upon examples from in vitro models that focused on M. tuberculosis and L. donovani, we review evidence that activation of host cell phosphotyrosine phosphatases may contribute to pathogenesis. A leading candidate appears to be the Src homology 2 domain containing phosphotyrosine phosphatase SHP-1, the activation of which may contribute to the development of infection and disease progression.

Phosphorylation of a gelatin-binding protein from L6 myoblasts by protein kinase C.

A gelatin-binding glycoprotein from L6 rat myoblasts, designated gp46, was shown to be phosphorylated in vivo. This phosphorylation was increased slightly (18%) by phorbol ester treatment of L6 suggesting protein kinase C involvement. Purified gp46 could be phosphorylated in vitro with protein kinase C, but not by the catalytic subunit of cAMP-dependent protein kinase. Comparison of the phosphotryptic peptide maps of in vitro and in vivo labeled gp46 suggested that in vivo phosphorylation of gp46 may be mediated by protein kinase C.

Bacterial and host-derived cationic proteins bind alpha2-laminins and enhance Mycobacterium leprae attachment to human Schwann cells.

It has recently been demonstrated that laminin alpha2 chains present on the surface of Schwann cells are involved in the process of attachment of Mycobacterium leprae to these cells. In this study, a protein in the M. leprae cell wall that was found to be capable of binding alpha2-containing laminins (merosin) was isolated and characterized. The M. leprae laminin-binding protein was identified as a 21-kDa histone-like protein (Hlp), a highly conserved cationic protein present in other species of mycobacteria. The gene that encodes this protein was PCR amplified, cloned, and expressed, and the recombinant protein was shown to bind alpha2-laminins. More significantly, when added exogenously, Hlp was able to greatly enhance the attachment of mycobacteria to ST88-14 human Schwann cells. The capacity to bind alpha2-laminins and to enhance mycobacterial adherence to Schwann cells was also found in other cationic proteins such as host-derived histones. Moreover, mutation in the hlp gene was shown not to affect the capacity of mycobacteria to bind to ST88-14 cells, suggesting that alternative adhesins and/or pathways might be used by mycobacteria during the process of adherence to Schwann cells. The potential role of Hlp as a fortuitous virulence factor contributing to the pathogenesis of M. leprae-mediated nerve damage is discussed.

A rapid, single-step purification method for immunogenic members of the hsp 70 family: validation and application.

Gelatin affinity chromatography has been developed as a simple one-step procedure for purification of members of the hsp 70 kDa family from MDBK cells (a bovine epithelioid cell line), rat liver microsomes and three different protozoan parasites. The ability of the isolated proteins to bind to denatured proteins like gelatin together with their apparent molecular masses, constitutive and inducible expression and their release from gelatin-agarose beads by ATP suggested that these proteins are molecular chaperones. The identity of a gelatin bound, ATP released, 78 kDa protein isolated from rat liver was confirmed by comparison of its NH2-terminal sequence with that of grp 78/BiP from rat. A 68 kDa protein isolated from Trypanosoma brucei brucei (T.b. brucei) and proteins of 68 and 69 kDa from Leishmania donovani (L. donovani) using gelatin affinity chromatography reacted in Western blot analysis with a monoclonal antibody, 7.10, specific for members of the 70 kDa heat shock protein family derived from a wide variety of species. A different monoclonal antibody, SPA-820, which also recognises members of the hsp 70 kDa family, bound to proteins isolated from Theileria parva Muguga transformed lymphoblastoid cell lines (TpM). The gelatin bound ATP released proteins of 72 kDa from T.b. brucei and of 65, 69 and 72 kDa from TpM were detected by recovery sera of the cattle infected with T.b. brucei and T. parva, respectively.

Delivery practices in west Uttar Pradesh.

The present study has been planned to identify healthy and unhealthy delivery practices in rural U.P. specially with reference to five cleans recommended under national C.S.S.M. programme. A total 120 women who had delivered in recent past were interviewed. Only in 3.1 percent deliveries, proper washing of floor was done, in 43 percent deliveries the cord cutting instrument was not sterilized and in about 65 percent deliveries mustard oil and ghee was used as first cord applicant.

PIP: Infant mortality remains high in the northern states of India. Neonatal tetanus is one of the important causes of early infant death in the region. The authors identified healthy and unhealthy delivery practices in rural West Uttar Pradesh based upon interview data from 120 women who recently delivered at home. 98 of the deliveries occurred in the household living room, while the remaining 22 were conducted elsewhere in the home. The floor was properly washed in only 3.1% of deliveries, the cord cutting instrument was not sterilized in 43% of deliveries, and mustard oil and deshi ghee were used as first cord applicants in approximately 65% of deliveries. Hygienic delivery practices and settings are not commonly used in this study area. Trained birth attendants were no better aware of cleanliness in delivery practice than untrained birth attendants. Community awareness was also lacking. It is recommended that birth attendants have periodic refresher training and a viable monitoring system of birth attendant-conducted deliveries be established.

Some bio-social factors for drop out of children undergoing oral polio vaccination.

PIP: This study was designed to identify biosocial factors that contribute to India's high dropout rate for completion of the oral polio vaccine series. The study sample included the 2027 children registered in 1983-84 at an immunization clinic in Agra. At the time of the 1st dose, parents were educated about the importance of completing the series. 683 of the children were not returned for the 3rd dose, for a dropout rate of 33.7%. The majority of dropouts (42.6%) were clustered in the 3-6 month age group. There was a higher but nonsignificant dropout rate in females (35.4%) than in males (32.6%). The dropout rate was lowest among children of birth order 1 (23.2%) and highest in birth order 5 (72.1%). There was a statistically significant association between dropout rates and maternal occupational status; only 3.8% of children whose mothers were engaged in service occupations failed to complete the immunization series compared with 34.6% of those whose mothers were housewives. In addition, there was a significant association with paternal occupation. The maximum number of dropouts was noted among children whose fathers were laborers (51.2%) and the lowest rate was found among children of men in service occupations (23.6%). Finally, there was a significant inverse relationship between parental education and dropout rates. This relationship was most pronounced for maternal educational status. The dropout rate was only 5% in children whose mothers were educated at the high school level and above compared with 14% in children whose father's education was at this level.^ieng

Reproductive tract infection in women of reproductive age group in Sitapur/Shahjahanpur District of Uttar Pradesh.

CARE India with its UP Office has initiated demand generation and acceptability of services for reproductive health and birth spacing services at community level in two districts in the state of Uttar Pradesh. A total of 3,49,000 women of reproductive age will benefit from participation in program activities over the next five years. Out of total surveyed population of 3662, 798 women of reproductive age group were identified of whom 272 were found to be symptomatically positive in relation of STDs/RTIs giving the overall prevalence rate of 34%. Clinical as well as the microbiological examination was carried out on 193 of these 272 women in field conditions. Those found clinically and/or microbiologically positive were provided treatment and preventive advice. Total of 150 (77.7%) women were diagnosed on the basis of symptoms (syndromic approach) and 151 (78.2%) were diagnosed on the basis of clinical examinations. However, 137 (70.9%) were confirmed microbiologically positive. A large extent was found in the age group of 15 to 24 years. Confirmed cases were comparatively less among nulliparous women (63.6%) than multipara (73.2%) cases. 64.1% women were having watery discharge while 29.1% were having complaint of curd discharge. The discharge was offensive in only 1.9% cases while 4.9% reported mixed discharge. 54.4% women complaining of vaginal discharge were suffering from PID. The discharge were either bacterial alone (26.1%) or bacterial mixed with candidiasis (16.5%) or trichomoniasis (8.7%). Compared to syndromic approach, an improvement in the sensitivity (81.8%) and predictive accuracy (74.1%) was noted when cases were detected with the help of clinical examination.

Role of traditional healers and indigenous medical practitioners in health care.

Nearly three-fourth of the indigenous medical practitioners (74.37%) in the rural area of Agra were treating 15 patients in a day. Maximum number of patients was attended by trained practitioners. Only 21.82 percent of the practitioners were providing preventive services apart from curative services. Inspite of wide variations in level of training with consequent difference in knowledge, skills and practice, these practitioners still make a significant contribution to health care of the community provided they get some training of modern health system and state patronage. To start with, at least they should be given orientation towards vaccination as they are still giving tetanus toxoid only at the time of injury. In the same way training should be given regarding distribution of vitamin 'A' for prevention of night blindness and iron folic acid tablets for control of anaemia in vulnerable groups.

Fever of unknown origin: a case of post obstructive pneumonia complicating mature teratoma.

Mediastinal teratomas are rare germ cell tumors in children accounting for only 4.3% of all germ cell tumours. Here, we describe a three year old child who was misdiagnosed as a case of pulmonary tuberculosis at periphery despite of his chest X ray showing large homogenous opacification of left hemithorax with areas of calcifications and subsequently diagnosed as a case of benign mature teratoma with post obstructive pneumonia. Our case highlights the need for careful evaluation of chest X-ray (CXR) by the treating physicians, especially when CXR had a large homogenous opacity with shifting of mediastinum and presence of a few calcified shadows, which may clinch a rare diagnosis of an uncommon disorder.

Primary neuroendocrine mediastinal tumor presenting with carcinoid syndrome and left supraclavicular lymphadenopathy: clinico-radiological and pathological features.

Primary mediastinal neuro-endocrine tumor is very rare. The primary modality to evaluate the lesion is computed tomography, to know disease extent, involvement of various structures, vascular invasion and metastasis. Histo-pathological and immuno-histochemical confirmation is mandatory. We report a rare case of primary neuroendocrine mediastinal tumor/atypical carcinoid in a young male who presented with carcinoid syndrome and left supraclavicular lymphadenopathy. Complete diagnostic work up was done followed by histo-pathological and immuno-histochemical confirmation. Later on patient underwent radical surgery followed by chemotherapy. The patient is currently on follow up. Neuroendocrine carcinoma of the thymus generally follows an aggressive clinical course.The biologic behavior is directly related to grade and degree of differentiation. This case report of primary low grade neuroendocrine tumor/atypical carcinoid adds to the biological behavior of this tumor and sheds light on the radiological and pathological features of neuroendocrine carcinomas.

A rare case of Balo concentric sclerosis showing unusual clinical improvement and response with oral prednisolone.

The present report is a rare case of Balo Concentric Sclerosis. Most cases have either been diagnosed post mortem or have succumbed to the disease after being diagnosed ante mortem. In our case, the patient showed a dramatic response to treatment, and after a one-year follow-up, he was asymptomatic, with no relapses or residual effect of the illness.

Potential role of ultrasound in anesthesia and intensive care.

One of the most exiting recent technological advances in the field of anesthesia to track the region of interest is the introduction of anatomical evaluation by ultrasound imaging. Widespread use of this modality depends on its proven clinical efficacy, cost effectiveness, and practicality as it allows anesthesiologist to evaluate complex and varied anatomy prior to needle insertion. Sound used in medicine is not significantly transmitted by air or bone but through fluids which make up the larger part of soft tissues in the body. Ultrasound has been shown to offer excellent guidance for difficult venous access, epidural space identification in cases of difficult anatomy, delineating nerve plexuses for chronic nerve blocks, for regional anesthesia, and in transesophageal echocardiography for cardiac imaging with blood flows or in an otherwise high-risk patient where interventional procedure is required. It has special application to assess the narrowest diameter of the subglottic upper airway. A systemic literature search was performed in PubMed and the Cochrane library. The search strategy was set up using either single text word or combinations. We also included the studies where in these techniques were compared with conventional methods . Despite the initial excitement of this technique, ultrasound visualization is still indirect and images are subject to individual interpretation. It is gradually becoming routine in daily practice at our institution due to its reliability and safety. Though ultrasound is much safer, exposure in terms of intensity and time should be limited as far as possible, as high-energy ultrasound can cause heating and damage to tissues. In this review, we discuss established and future areas of ultrasound imaging and emphasize the use of B-mode ultrasound to improve the efficacy of interventional techniques. We have also illustrated potential uses with reference to cross-sectional B-mode images which visually represent a slice of tissues and are the easiest images for interpretation by clinicians.