Human tissue kallikrein 9: production of recombinant proteins and specific antibodies.

Human tissue kallikreins (genes, KLKs; proteins, hKs) are a subgroup of hormonally regulated serine proteases. Two tissue kallikreins, namely hK2 and hK3 (prostate-specific antigen, PSA), are currently used as serological biomarkers of prostate cancer. Human tissue kallikrein 9 (KLK9) is a newly identified member of the tissue kallikrein gene family. Recent reports have indicated that KLK9 mRNA is differentially expressed in ovarian and breast cancer and has prognostic value. Here, we report the production of recombinant hK9 (classic form) using prokaryotic and mammalian cells and the generation of polyclonal antibodies. Total testis tissue mRNA was reverse-transcribed to cDNA, amplified, cloned into a pET/200 TOPO plasmid vector, and transformed into E. coli cells. hK9 was purified and used as an immunogen to generate polyclonal antibodies. Full-length KLK9 cDNA was also cloned in the vector pcDNA3.1 and was expressed in CHO cells. The identity of hK9 was confirmed by mass spectrometry. hK9 rabbit antiserum displayed no cross-reactivity with other tissue kallikreins and could specifically recognize E. coli- and CHO-derived hK9 on Western blots. hK9 was mainly detected in testis and seminal vesicles by Western blotting. The reagents generated here will help to define the physiological role of this tissue kallikrein and its involvement in human disease.

Iodine status remains critical in mother and infant in Central Anatolia (Kayseri) of Turkey.

BACKGROUND: Severe iodine deficiency disorders have been eradicated in many parts of the world, but milder forms still exist and may escape detection. Turkey has long been known to be a mild to moderate iodine deficiency area. AIM OF THE STUDY: The aim of this study was to assess the iodine nutritional status and the thyroid function of pregnant women and their neonates in the region of Kayseri (central Anatolia of Turkey) that appeared to be iodine deficient in previous studies performed before the introduction of mandatory salt iodization. METHODS: A cross-sectional voluntary screening study was performed in the Maternity Unit of a university hospital. A total of 70 mothers and their healthy full-term neonates were included in this study. Urinary iodine concentration was estimated in spot urine samples obtained from mothers and their neonates on day 5. All the neonates were breastfed. The iodine content was determined in the breast milk of all mothers on day 5. Serum concentrations of TSH, thyroglobulin (Tg), free triiodothyronine (FT3) and free thyroxine (FT4) were investigated in the cord serum of neonates and compared to those of mothers immediately after parturition RESULTS: The median urinary iodine on day 5 in mothers and their babies were 30.20 and 23.80 microg/l, respectively. These figures are much lower than normal for these age groups (150-200 microg/l). The median iodine content of breast-milk was 73 microg/l. It is again much lower than in iodine sufficient areas, indicating that the status of iodine nutrition of pregnant and lactating women is clearly insufficient. The median concentrations (and ranges) of neonatal TSH, Tg, FT3 and FT4 were 7.44 mU/l, 71.62 ng/ml, 1.30 pg/ml and 1.34 ng/dl respectively. The corresponding levels for the mothers during labor were 2.19 mU/l, 25.65 ng/ml, 1.31 pg/ml and 1.23 ng/dl respectively. The median neonatal serum concentrations of TSH and Tg were significantly higher than the corresponding maternal levels (P < 0.0001, P < 0.0001, respectively) and 27.1% of the neonates had serum TSH concentrations above 10 mU/l and 57.1 % had cord blood serum Tg concentrations above 54 ng/ml. None of the mothers showed TSH concentrations above 5 mU/l and 41.4% had serum Tg concentrations above 30 ng/ml. CONCLUSION: Iodine deficiency with low urinary iodine excretion and high serum Tg and TSH concentrations were recognized among pregnant women and their babies in Kayseri in spite of the program of salt iodization. National measures are urgently required for improving the correction of iodine deficiency in Turkey. This includes regular supplementation with iodine, starting at preconception or in early pregnancy and continuing during the period of nursing in this region.

Iodine deficiency in pregnant women and in their neonates in the central Anatolian region (Kayseri) of Turkey.

Severe iodine deficiency disorders may have been eradicated in many parts of the world, but milder forms still exist and may escape detection. The aim of this study was to assess the iodine nutritional status of pregnant women and their newborns, and the prevalence rates and severity of iodine deficiency in the Kayseri region, which has appeared to be iodine deficient in previous studies. A cross-sectional voluntary screening study was performed in the Maternity Unit of the University Hospital. Seventy pregnant women and their babies participated in this study. Iodine deficiency with high prevalence of goiter, low urinary iodine excretion and high serum thyroglobulin concentrations were recognized among pregnant women and their babies in Kayseri. Regular administration of iodine, starting at preconception or in early pregnancy and continuing during the period of nursing, is recommended in these regions.

Use of olive oil based emulsions as an alternative to soybean oil based emulsions in total parenteral nutrition and their effects on liver regeneration following hepatic resection in rats.

BACKGROUND/AIM: The main objective of this study was to compare the effects of olive oil to those of soybean oil on liver tissue regeneration following hepatic resection in rats. METHODS: Seventy albino Wistar rats were randomly assigned to seven groups which contained 10 rats each. Group 1 was the sham-treated group; groups 2 and 3 received total parenteral nutrition (TPN) containing soybean oil lipid emulsions (20% Lipofundin MCT/LCT) for 48 or 72 h; groups 4 and 5 received TPN containing olive oil (80%)/soybean oil (20%) lipid emulsions (ClinOleic 20%) for 48 or 72 h; group 6 was the control group for 48 h, and group 7 was the control group for 72 h. TPN was given via internal jugular vein, and 70% hepatic resection was performed in the study groups. In addition, hepatic resections with no TPN were performed in the control groups, except the sham group. Relative liver weight, mitotic index, proliferating cell nuclear antigen labeling index, and carnitine levels in liver tissue samples were used to assess hepatic regeneration. Thiobarbituric acid reactive substances were measured as an index of lipid peroxidation and oxidative tissue damage. Alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase were parameters for the liver function. RESULTS: The relative liver weight increased significantly with minimal hepatosteatosis in the olive oil treated groups. Lipid peroxidation significantly decreased with near-normal serum levels of the liver function parameters in the olive oil/soybean oil treated groups, and mitotic index and proliferating cell nuclear antigen labeling index reached their maximum level in rats receiving TPN containing any kind of lipid emulsions for 48 h following resection. CONCLUSIONS: The use of olive oil/soybean oil lipid emulsions in TPN has important beneficial effects on the liver regeneration, and the antioxidant properties of olive oil originating from its natural components indirectly contribute to the liver regeneration in rats.

Nitric oxide levels in tears of patients with mild forms of papillary conjunctivitis induced by rigid gas-permeable contact lenses.

PURPOSE: To determine whether nitric oxide (NO) is detectable in the tear fluid of patients with mild forms of papillary conjunctivitis who wear rigid gas-permeable contact lenses. METHODS: Tear samples were taken from 12 users of rigid gas-permeable contact lenses and 12 healthy controls. Samples were analyzed, and the levels of NO were determined. RESULTS: The mean level of NO in those who wore contact lenses was 118.98 micromol/L (95% CI: 91.85-146.10 micromol/L); whereas the level of NO found in the control subjects was 114.84 micromol/L (95% CI: 102.54-127.14 micromol/L). The difference was statistically insignificant (P>0.05). CONCLUSION: Nitric oxide levels in the tear fluid of rigid gas-permeable contact lens users did not correlate with the presence of mild symptoms of papillary conjunctivitis.

The effect of folic acid, vitamin B6 and vitamin B12 on the homocysteine levels in rabbits fed by methionine-enriched diets.

Atherosclerosis is an important cause of cardiovascular morbidity and mortality in recent years. Hyperhomocysteinemia is recognized as an independent risk factor for premature atherosclerosis and venous thrombosis. It is suggested that administration of folic acid, vitamin B6 and vitamin B12 may decrease homocysteine levels. In our study, we induced hyperhomocysteinemia in rabbits by giving methionine and studied the effects of folic acid, vitamin B6 and vitamin B12 on homocysteine levels. A total of 40 (20 female, 20 male New Zealand rabbits) were divided into four groups, each consisting of 10 rabbits. Methionine (100 mg/kg/day), methionine (100 mg/kg/day) plus vitamin B6 (30 mg/kg/day), methionine (100 mg/kg/day) plus vitamin B12 (80 mg/kg/day) and methionine (100 mg/kg/day) plus folic acid (20 mg/kg/day) were given to the first, second, third and forth groups respectively. These rabbits were followed up for two months. We studied homocysteine levels on the 0, 20th, 40th and 60th days in all groups. In rabbits we induced hyperhomocysteinemia by giving methionine for 2 months. The decreases of homocysteine levels in the forth group were significant with respect to the second and third groups. Folic acid supplementation clearly resulted in a reduction of plasma homocysteine levels, whereas vitamin B12 was little effective and vitamin B6 failed to show an effect. We conclude that even folic acid treatment alone may be sufficient for decreasing negative effects of homocysteine.