Visualization of corticotropin-releasing factor neurons by fluorescent proteins in the mouse brain and characterization of labeled neurons in the paraventricular nucleus of the hypothalamus.

Corticotropin-releasing factor (CRF) is the key regulator of the hypothalamic-pituitary-adrenal axis. CRF neurons cannot be distinguished morphologically from other neuroendocrine neurons in the paraventricular nucleus of the hypothalamus (PVH) without immunostaining. Thus, we generated a knock-in mouse that expresses modified yellow fluorescent protein (Venus) in CRF neurons (CRF-Venus), and yet its expression is driven by the CRF promoter and responds to changes in the interior milieu. In CRF-Venus, Venus-expressing neurons were distributed in brain regions harboring CRF neurons, including the PVH. The majority of Venus-expressing neurons overlapped with CRF-expressing neurons in the PVH, but many neurons expressed only Venus or CRF in a physiological glucocorticoid condition. After glucocorticoid deprivation, however, Venus expression intensified, and most Venus neurons coexpressed CRF. Conversely, Venus expression was suppressed by excess glucocorticoids. Expression of copeptin, a peptide encoded within the vasopressin gene, was induced in PVH-Venus neurons by glucocorticoid deprivation and suppressed by glucocorticoid administration. Thus, Venus neurons recapitulated glucocorticoid-dependent vasopressin expression in PVH-CRF neurons. Noradrenaline increased the frequency of glutamate-dependent excitatory postsynaptic currents recorded from Venus-expressing neurons in the voltage clamp mode. In addition, the CRF-iCre knock-in mouse was crossed with a CAG-CAT-EGFP reporter mouse to yield the Tg(CAG-CAT-EGFP/wt);CRF(iCre/wt) (EGFP/CRF-iCre) mouse, in which enhanced green fluorescent protein (EGFP) is driven by the CAG promoter. EGFP was expressed more constitutively in the PVH of EGFP/CRF-iCre mice. Thus, CRF-Venus may have an advantage for monitoring dynamic changes in CRF neurons and CRF networks in different glucocorticoid states.

[Case of bladder tuberculosis with onset at the age of nineteen--treatment of urinary tract tuberculosis in accordance with the new Japanese Tuberculosis Treatment Guidelines].

A 24-year-old man experienced gross haematuria and dysuria several times a year from the age of 19, presenting to this Department for the first time at age 21, when he was given standard antibiotic treatment for acute cystitis. Although urinary symptoms persisted, he failed to attend for follow-up. He attended another clinic at the age of 24 with increased urinary frequency. Transrectal ultrasonography revealed thickening of the bladder wall, concavity of the right bladder neck, and nodular changes extending from the left bladder neck to the left bladder wall, so he was referred to this department for further investigation. Mycobacterium tuberculosis was detected in the urine by the referring doctor, so the diagnosis was made of bladder tuberculosis (TB). We treated him with rifampicin (RFP), isoniazid (INH) and pyrazinamide (PZA) triple therapy for 2 months, followed by RFP and INH dual therapy for 4 months. His urinary frequency improved markedly after one month, and his bladder capacity was 420 ml after 4 months of treatment. After 2 and half year follow-up he remains well without any signs of relapse. To our knowledge, this is only the ninth case of teenage onset of urinary tract TB in Japan since 1995. As specified in Clause 22 of the Enforcement Regulations of the Tuberculosis Control Law, chemotherapy and surgical treatment of TB, the mainstays of treatment, should be administered in accordance with the 'Standards for the Treatment of Tuberculosis', issued by the Japanese Minister of Health and revised in 2004. The level of recognition of the 'Standards for the Treatment of Tuberculosis' is low, however. Although the incidence of TB of the urinary tract has dropped dramatically, as urologists we must be aware that treatment of this condition must be given in accordance with the Standards.

Extraskeletal mesenchymal chondrosarcoma of the kidney.

A 61-year-old woman was referred for evaluation of an incidental mass found on the right kidney. Abdominal ultrasonography and computed tomography showed a 3-cm tumor with calcification and peripheral enhancement. Under the diagnosis of renal cell carcinoma, a transabdominal right radical nephrectomy was performed. The pathological diagnosis was extraskeletal mesenchymal chondrosarcoma of the kidney. No recurrence or metastasis occurred during a 6-year follow up. Primary renal chondrosarcoma is extremely rare, this being only the fifth case reported in the English literature, and the patient's extended survival is attributed to the small size of the tumor, as well as early diagnosis and treatment.

Isolation and partial characterization of fucan sulfates from the body wall of sea cucumber Stichopus japonicus and their ability to inhibit osteoclastogenesis.

Two types of fucan sulfate were isolated from chloroform/methanol extract of the body wall of the sea cucumber Stichopus japonicus. One type (type A) contained 3.41 mmol fucose/g and 2.35 mmol sulfate/g, and the molecular mass was determined to be 9 kDa by gel permeation chromatography (GPC). Structural analysis suggested that type A consists of a backbone of (1-->3)-linked fucosyl residues that are substituted at C-4 with fucosyl residues, and that fucosyl residues are sulfated at C-2 and/or C-4. Another type (type B) contained 3.90 mmol fucose/g and 3.07 mmol sulfate/g, and the molecular mass was determined to be 32kDa by GPC. Structural analysis showed that type B is largely composed of unbranched (1-->3)-linked fucosyl residues, and that sulfate substitution(s) occur at C-2 and/or C-4. The potential of both types to inhibit osteoclastogenesis was examined by an in vitro assay system, showing that both types of fucan sulfate inhibit osteoclastogenesis more than 95% at 50 microg/mL concentration. These results suggest that types A and B fucan sulfate from sea cucumber are potent inhibitors of osteoclastogenesis.

High-resolution crystal structure of Arthrobacter aurescens chondroitin AC lyase: an enzyme-substrate complex defines the catalytic mechanism.

Chondroitin lyases (EC 4.2.2.4 and EC 4.2.2.5) are glycosaminoglycan-degrading enzymes that act as eliminases. Chondroitin lyase AC from Arthrobacter aurescens (ArthroAC) is known to act on chondroitin 4-sulfate and chondroitin 6-sulfate but not on dermatan sulfate. Like other chondroitin AC lyases, it is capable of cleaving hyaluronan. We have determined the three-dimensional crystal structure of ArthroAC in its native form as well as in complex with its substrates (chondroitin 4-sulfate tetrasaccharide, CS(tetra) and hyaluronan tetrasaccharide) at resolution varying from 1.25 A to 1.9A. The primary sequence of ArthroAC has not been previously determined but it was possible to determine the amino acid sequence of this enzyme from the high-resolution electron density maps and to confirm it by mass spectrometry. The enzyme-substrate complexes were obtained by soaking the substrate into the crystals for varying lengths of time (30 seconds to ten hours) and flash-cooling the crystals. The electron density map for crystals soaked in the substrate for as short as 30 seconds showed the substrate clearly and indicated that the ring of central glucuronic acid assumes a distorted boat conformation. This structure strongly supports the lytic mechanism where Tyr242 acts as a general base that abstracts the proton from the C5 position of glucuronic acid while Asn183 and His233 neutralize the charge on the glucuronate acidic group. Comparison of this structure with that of chondroitinase AC from Flavobacterium heparinum (FlavoAC) provides an explanation for the exolytic and endolytic mode of action of ArthroAC and FlavoAC, respectively.

[Transcatheter arterial embolization for bleeding of prostatic artery after prostate biopsy].

We present a case of bleeding from the prostatic artery, complicating transrectal ultrasound (TRUS) guided prostate needle biopsy, that responded to transcatheter arterial embolization (TAE). A 62-year-old man with a serum PSA of 4.1 ng/ml was admitted to this institution for a prostate biopsy. He developed hypotension and marked abdominal distension 3 hours after undergoing TRUS guided prostate needle biopsy. CT scanning revealed a massive hematoma extending from the pelvis into the retroperitoneal space. Intra-arterial digital subtraction angiography (IA-DSA) showed extravasation of dye from the right prostatic artery, indicating that it had been damaged during the biopsy procedure. The bleeding was successfully stopped with TAE, using 6 micro coils. TRUS guided prostate biopsy is generally considered a safe procedure, with few complications, and cases of massive hemorrhage into the retroperitoneal space are extremely rare. In cases of arterial retroperitoneal bleeding such as this one, treatment with TAE is fast and accurate.

Enhancement of t-PA-mediated plasminogen activation by partially defucosylated glycosaminoglycans from the sea cucumber Stichopus japonicus.

Sea cucumber glycosaminoglycan (SC-GAG) was isolated from the body wall of the sea cucumber Stichopus japonicus. The SC-GAG consists of a chondroitin sulfate E-type core polymer with sulfated fucose branches attaching glycosidically to almost every disaccharide unit of the core polymer at the C-3 position of the GlcA or at C-4 and/or C-6 position(s) of GalNAc. SC-GAG was subjected to mild acid-hydrolysis, which cleaved selectively the glycosidic linkages between the core polymer and the fucose branches, resulting in two types of partially defucosylated SC-GAG derivatives. One type (type A), obtained by 3 h-hydrolysis, contained 33% of the fucose branches and the other type (type B), obtained by 6-h hydrolysis, contained 10% of the fucose branches. The molecular masses of types A and B were determined to be 8 and 4 kDa, respectively, by gel permeation HPLC. A chondroitinase ABC (Chase ABC)-digestion demonstrated that types A and B contained 46 and 66% of digestable disaccharide units, respectively, and both types contained 29% of E-type unsaturated disaccharide units bearing no fucose branches. Intact SC-GAG and types A and B were compared for t-PA-mediated plasminogen activation by an in vitro assay system. Although intact SC-GAG and type B exhibited rather weak activity at 6.25 microg/ml, type A exhibited 5 to 10-fold higher activity than intact SC-GAG and type B at the same concentration. The activity of type A was almost one-third that of purified chondroitin sulfate E (127 kDa containing 64.5% E-type disaccharide units) from squid cartilage at 6.25 microg/ml concentration. These results suggest that t-PA-mediated plasminogen activation requires the presence of E-type disaccharide units bearing no fucose branches and a molecular mass larger than 7.5 kDa in terms of the chondroitin sulfate E structure with or without fucose branching.

[The use of predeposited autologous blood transfusion for radical prostatectomy].

PURPOSE: In reference to the cases in which radical prostatectomy was performed after storage of autologous blood, we retrospectively study the usefulness of this procedure and proper amount of blood stored. PATIENTS AND METHODS: The subjects included 62 cases in which radical prostatectomy was performed after storage of autologous blood from October, 1997 to March, 2000. As the amount of blood to be stored, either 800 ml or 1,200 ml was selected optionally as the amount of blood to be stored, and blood was stored at a rate of 400 ml once a week. Erythropoietin, 24,000 units was injected subcutaneously after storage of blood, and an iron preparation 200 mg/day was administered orally throughout the period. RESULTS: Homologous blood transfusion could be avoided in 58 out of 62 cases, the avoidance rate being 93.5%. With 200 ml of autologous blood as 1 unit, 104 units out of 330 units were discarded, the disposal rate being 31.5%. To lower the disposal rate, we studied whether there is any parameter that can predict the loss of blood preoperatively. As a result, we found a significant difference in the loss of blood between the body mass index of less than 24 and that of more than 24. Blood storage and transfusion produced no side-effects. CONCLUSION: Storing autologous blood in radical prostatectomy is considered useful since homologous blood transfusion can be avoided at a high percentage and no side-effects are produced. The body mass index is useful for predicting the loss of blood and determining a proper amount of blood to be stored.