Protein inhibitor of nitric oxide synthase (NOS) and the N-methyl-D-aspartate receptor are expressed in the rat and mouse penile nerves and colocalize with penile neuronal NOS.

Nitrergic neurotransmission triggering penile erection is mediated by nitric oxide (NO) synthesized in the cavernosal nerves of the penis by penile neuronal NO synthase (PnNOS). In the central nervous system, nNOS is activated by the N-methyl-D-aspartate receptor (NMDAR) and, presumably, is inhibited by the protein inhibitor of NOS (PIN). The PnNOS and NMDAR are expressed in the penis, and PnNOS has been localized in penile nerves. Both proteins colocalize with PIN in the hypothalamus and the spinal cord involved in the control of erection. The present study aimed to elucidate the relationship between PnNOS, PIN, and NMDAR in the penis. It was found that in the rat, PIN was expressed in the pelvic ganglion and the cavernosal nerve, and penile PIN cDNA was cloned, sequenced, and expressed. Immunohistochemistry localized PIN to the cavernosal and dorsal nerve of the penis, whereas NMDAR was not detected in the latter. Dual-fluorescence labeling showed that PnNOS colocalized with PIN in both nerves but with NMDAR only in the cavernosal nerve. Aging did not affect the mRNA levels of PnNOS, nNOS, NMDAR, and PIN. Both PIN and NMDAR were detected in penile nerves of the wild-type and nNOS(-/-) mouse. The PIN protein did not inhibit or bind NOS in penile extracts, and in vivo, PIN cDNA reduced the erectile response to electrical field stimulation. In conclusion, PIN and NMDAR colocalize with PnNOS in penile nerves, but the functional significance of these protein interactions for penile erection remains to be elucidated.

Myostatin and insulin-like growth factor-I and -II expression in the muscle of rats exposed to the microgravity environment of the NeuroLab space shuttle flight.

The mechanism of the loss of skeletal muscle mass that occurs during spaceflight is not well understood. Myostatin has been proposed as a negative modulator of muscle mass, and IGF-I and IGF-II are known positive regulators of muscle differentiation and growth. We investigated whether muscle loss associated with spaceflight is accompanied by increased levels of myostatin and a reduction in IGF-I and -II levels in the muscle, and whether these changes correlate with an increase in muscle proteolysis and apoptosis. Twelve male adult rats sent on the 17-day NASA STS-90 NeuroLab space flight were divided upon return to earth into two groups, and killed either 1 day later (R1) or after 13 days of acclimatization (R13). Ground-based control rats were maintained for the same periods in either vivarium (R3 and R15, respectively), or flight-simulated cages (R5 and R17, respectively). RNA and protein were isolated from the tibialis anterior, biceps femoris, quadriceps, and gastrocnemius muscles. Myostatin, IGF-I, IGF-II and proteasome 2c mRNA concentrations were determined by reverse transcription/PCR; myostatin and ubiquitin mRNA were also measured by Northern blot analysis; myostatin protein was estimated by immunohistochemistry; the apoptotic index and the release of 3-methylhistidine were determined respectively by the TUNEL assay and by HPLC. Muscle weights were 19-24% lower in the R1 rats compared with the control R3 and R5 rats, but were not significantly different after the recovery period. The myostatin/beta-actin mRNA ratios (means+/-s.e.m. ) were higher in the muscles of the R1 rats compared with the control R5 rats: 5.0-fold in tibialis (5.35 +/- 1.85 vs 1.07 +/- 0.26), 3.0-fold in biceps (2.46+/-0.70 vs 0.81 +/- 0.04), 1.9-fold in quadriceps (7.84 +/- 1.73 vs 4.08 +/- 0.52), and 2.2-fold in gastrocnemius (0.99 +/- 0.35 vs 0.44 +/- 0.17). These values also normalized upon acclimatization. Our antibody against a myostatin peptide was validated by detection of the recombinant human myostatin protein on Western blots, which also showed that myostatin immunostaining was increased in muscle sections from R1 rats, compared with control R3 rats, and normalized upon acclimatization. In contrast, IGF-II mRNA concentrations in the muscles from R1 rats were 64-89% lower than those in R3 animals. With the exception of the gastrocnemius, IGF-II was also decreased in R5 animals maintained in flight-simulated cages, and normalized upon acclimatization. The intramuscular IGF-I mRNA levels were not significantly different between the spaceflight rats and the controls. No increase was found in the proteolysis markers 3-methyl histidine, ubiquitin mRNA, and proteasome 2C mRNA. In conclusion, the loss of skeletal muscle mass that occurs during spaceflight is associated with increased myostatin mRNA and protein levels in the skeletal muscle, and a decrease in IGF-II mRNA levels. These alterations are normalized upon restoration of normal gravity and caging conditions. These data suggest that reciprocal changes in the expression of myostatin and IGF-II may contribute to the multifactorial pathophysiology of muscle atrophy that occurs during spaceflight.

Organization of the human myostatin gene and expression in healthy men and HIV-infected men with muscle wasting.

Myostatin, a member of the transforming growth factor-beta superfamily, is a genetic determinant of skeletal muscle growth. Mice and cattle with inactivating mutations of myostatin have marked muscle hypertrophy. However, it is not known whether myostatin regulates skeletal muscle growth in adult men and whether increased myostatin expression contributes to wasting in chronic illness. We examined the hypothesis that myostatin expression correlates inversely with fat-free mass in humans and that increased expression of the myostatin gene is associated with weight loss in men with AIDS wasting syndrome. We therefore cloned the human myostatin gene and cDNA and examined the gene's expression in the skeletal muscle and serum of healthy and HIV-infected men. The myostatin gene comprises three exons and two introns, maps to chromosomal region 2q33.2, has three putative transcription initiation sites, and is transcribed as a 3.1-kb mRNA species that encodes a 375-aa precursor protein. Myostatin is expressed uniquely in the human skeletal muscle as a 26-kDa mature glycoprotein (myostatin-immunoreactive protein) and secreted into the plasma. Myostatin immunoreactivity is detectable in human skeletal muscle in both type 1 and 2 fibers. The serum and intramuscular concentrations of myostatin-immunoreactive protein are increased in HIV-infected men with weight loss compared with healthy men and correlate inversely with fat-free mass index. These data support the hypothesis that myostatin is an attenuator of skeletal muscle growth in adult men and contributes to muscle wasting in HIV-infected men.

Suggested reference ranges in clinical chemistry for apparently healthy males and females of Pakistan.

Seven hundred and eighty six apparently healthy males (418) and females (368) aged 0-69 years were randomly selected for estimation of reference ranges of 24 serum analytes at the clinical chemistry laboratory of The Aga Khan University Hospital (AKUH). Of the total study samples, 56% (439/786) were in the paediatric age group (0-14 years) and 44% (347/786) in the adult (15 > or = 60 years) group. Beckman Astra Ideal Autoanalyzer was used for all the estimations. Mean and standard deviations (SD) were calculated for each of the age groups. Reference ranges were calculated following standard methods of the International Federation of Clinical Chemistry (IFCC) and International Committee for Standardization in Haematology (ICSH).

Factors associated with elevated serum cholesterol levels in well-to-do Pakistani schoolchildren.

Cholesterol (CH) and triglyceride (TG) levels were determined in blood drawn after an overnight fast from 388 school children aged 5-19 years from private schools in Karachi. The mean CH levels ranged from 4.4 to 4.6 mmol l-1 (170.1 to 177.9 mg dl-1) for boys and 4.4 to 4.8 mmol l-1 (170.1 to 185.6 mg dl-1) for girls. The range of TG levels was 1.0 to 1.2 mmol l-1 (88.6 to 106.3 mg dl-1) and 0.9 to 1.1 mmol l-1 (79.7 to 97.4 mg dl-1) for boys and girls respectively. Sixty-two per cent of the girls and 54% of the boys had cholesterol values greater than or equal to 4.4 mmol l-1 (170 mg dl-1), a level at which dietary intervention is recommended for children. Thirty-two per cent of all the children had triglyceride levels above the 90th percentile of the levels for similar age groups in North America. The mean cholesterol intake was 469 mg/day for girls and 518 mg/day for boys. Overweight and inactivity were associated with raised serum cholesterol levels. Forty per cent of the girls and 25% of the boys reported a strong family history of hypercholesterolaemia and/or heart disease. The results show that the prevalence of hypercholesterolaemia is high in well-to-do Pakistani school children and factors which can be modified to lower serum cholesterol levels are identified.

Blood lipids in a healthy Karachi population.

Serum levels for cholesterol and triglycerides were estimated in an apparently normal healthy population of Karachi, aged between 4 and 59 years. In total, there were 632 subjects, 322 males and 310 females. Hypercholesterolemia was defined as a cholesterol level greater than 6.2 mmol l-1 (240 mg dl-1) in subjects above 20 years of age. In the age groups 20-39 and 40-59 years hypercholesterolemia was present in 26-41% of the males and 10-38% of the females. When triglyceride levels of more than 2.8 mmol l-1 (250 mg dl-1) were taken as abnormal for healthy males and females, 0-2% of the females and 10-25% of the males above 20 years of age were hypertriglyceridemic. The mean cholesterol levels in the age groups 4-9 and 10-19 years varied from 4.4 to 4.9 mmol l-1 (169.8 to 189.1 mg dl-1).

Serum alkaline phosphatase in apparently healthy Karachi population.

Serum alkaline phosphatase (AP) was estimated in a total of 786 (418 males and 386 females) apparently healthy people aged between 1-75 years selected randomly from a sample of the Karachi population. Reference ranges for AP level were obtained for the males and females stratified into ten successive age groups. The study population was also divided into two main age groups, a paediatric aged between 1-14 years and an adult group aged between 15 to over 50 years to see if the mean AP levels differ significantly between the two groups. Mean AP level for the male paediatric age group was 225 IU/L, significantly higher (P less than 0.005) than those of the male adult mean level of 83 IU/L. Similarly the mean AP levels for the paediatric female age group was 205 IU/L significantly higher (P less than 0.005), compared to the mean AP level of 67 IU/L obtained for the female adult age group.

Trace element studies on Karachi populations. Part V: Blood lead levels in normal healthy adults and grammar school children.

Blood lead levels of healthy Karachi population were estimated. Mean levels for males, females, soldiers and school children were 34.4, 31.8, 29.9 and 38.2 micrograms/dl respectively. About 93% cases of either sex had elevated lead levels, of whom 30% males and 10% females had levels above the safety limits (40 micrograms/dl). Soldiers living in relatively pollution free area though had levels lower than the rest of the population but 91% had levels over 25 micrograms/dl and only two had acceptable levels. Ninety-two percent children showed levels above 25 micrograms/dl with a large number having levels over 40 micrograms/dl. A very small percentage had normal levels. Pollution by traffic exhaust was assumed to be the principal cause for these high levels.

Serum cholesterol in neonates and their mothers. A pilot study.

Reports from the Aga Khan University indicate that 58% of 400 school children studied had undesirably high serum cholesterol levels. The present study was undertaken to determine whether the high cholesterol levels are present at birth and to determine the relationship between cord blood, maternal blood cholesterol and maternal diet. Cord blood from 58 neonates and fasting venous blood from 45 mothers were analyzed from total serum cholesterol. Mothers were interviewed regarding their usual diet during pregnancy. Mean cord blood cholesterol was 56.90 mg/dl (range 26 to 123 mg/dl). Mean maternal blood cholesterol was 232.4 mg/dl (range 141-382 mg/dl). Mean maternal intake of cholesterol was 457 mg (recommended level less than or equal to 300 mg/day). There was no significant co-relation between cord blood cholesterol and maternal blood cholesterol or maternal intake of cholesterol. Eighteen percent of the mothers reported a strong family history of hypercholesterolemia and/or heart disease, but this genetic tendency was not observed in the blood cholesterol level at birth indicating that environmental factors namely diet may have a prime role in determining serum cholesterol levels in childhood.

Efficacy of a home made quality control serum.

With increasing automation in Clinical Laboratories, the requirements for quality control material have greatly increased in order to monitor performance. The constant use of commercial control material is not economically feasible for many countries because of non-availability or the high cost of these materials. Here we describe a simple technique to prepare Home Made Quality Control serum using blood from polycythaemic patients. This preparation is stable for about six months without any alterations in the concentration of any of the chemical constituents. Extensive use of Home Made Quality Control sera in our laboratories have saved about 69% of the amount spent on the commercial material without any compromise in quality of the laboratory performance.

Trace element studies on Karachi population, Part II: Normal ranges for blood copper, zinc and magnesium for children and adolescents.

Normal ranges for copper, zinc and magnesium in whole blood have been established for healthy school going children of Indo-Pak origin. Age groups of 7 to 11 years had slightly elevated levels of these metals when compared with the groups below 7 and above 12 years, except that for females levels increased with age for zinc and decreased for magnesium.