Synthesis and application of caged peptides and proteins.

Caged compounds have covalently attached groups that are rapidly cleaved upon exposure to UV light. Attachment of photolabile groups makes the molecule inert until photolysis releases it in its bioactive form. When caged compounds are applied to the experimental system in advance, the concentration jump of biologically active substances can be brought about immediately in a limited area upon irradiation with pulsed and focused UV light. Therefore, caged compounds of low molecular weight, which are commercially available, have been used effectively to study the mechanisms of temporal biological phenomena, such as muscle contraction, intracellular signaling, and neurotransmission. Because many proteins and peptides play important roles in these phenomena, their caged derivatives should serve as powerful tools to clarify complex biological systems. To prepare caged proteins and peptides, several groups have improved upon a chemical modification method, as well as developed two new methods: (1) nonsense codon suppression and (2) solid-phase peptide synthesis. In this review, we summarize recent advances made in the design, preparation, and application of caged peptides and proteins.

Substrate specificity of Ca(2+)/calmodulin-dependent protein kinase phosphatase: kinetic studies using synthetic phosphopeptides as model substrates.

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKPase) dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases. In order to elucidate the mechanism of substrate recognition by CaMKPase, we chemically synthesized a variety of phosphopeptide analogs and carried out kinetic analysis using them as CaMKPase substrates. This is the first report using systematically synthesized phosphopeptides as substrates for kinetic studies on substrate specificities of protein Ser/Thr phosphatases. CaMKPase was shown to be a protein Ser/Thr phosphatase having a strong preference for a phospho-Thr residue. A Pro residue adjacent to the dephosphorylation site on the C-terminal side and acidic clusters around the dephosphorylation site had detrimental effects on dephosphorylation by CaMKPase. Deletion analysis of a model substrate peptide revealed that the minimal length of the substrate peptide was only 2 to 3 amino acid residues including the dephosphorylation site. The residues on the C-terminal side of the dephosphorylation site were not essential for dephosphorylation, whereas the residue adjacent to the dephosphorylation site on the N-terminal side was essential. Ala-scanning analysis suggested that CaMKPase did not recognize a specific motif around the dephosphorylation site. Myosin light chain phosphorylated by protein kinase C and Erk2 phosphorylated by MEK1 were poor substrates for CaMKPase, while a synthetic phosphopeptide corresponding to the sequence around the phosphorylation site of the former was not dephosphorylated by CaMKPase but that of the latter was fairly good substrate. These data suggest that substrate specificity of CaMKPase is determined by higher-order structure of the substrate protein rather than by the primary structure around its dephosphorylation site. Use of phosphopeptide substrates also revealed that poly-L-lysine, an activator for CaMKPase, activated the enzyme mainly through increase in the V(max) values.

Tc-99m PMT whole-body scintigraphy for evaluated of therapeutic effect and for monitoring bone metastasis in a patient with hepatocellular carcinoma.

Detection of metastatic lesions by bone scintigraphy is highly sensitive but has a low rate of specificity. Often bone metastases from hepatocellular carcinoma are not detected by bone scintigraphy because of low uptake or a photopenic area in the tumor. In contrast, Tc-99m Sn-N-pyridoxy-5-methyltryptophan (Tc-99m PMT) whole-body scintigraphy reflects tumor viability, and the specificity of detection is so high that tumor structure can be shown well. Tc-99m PMT whole-body scintigraphy was helpful for evaluating the response to therapy and monitoring the course of the patient described here with bone metastasis from hepatocellular carcinoma.

Serial changes in N-isopropyl-p[125I]-iodoamphetamine in mouse lung observed with a confocal laser scanning microscope.

Serial changes in N-isopropyl-p[125I]-iodoamphetamine (125I-IMP) in mouse lungs were observed with a confocal laser scanning microscope. Male mice were intravenously injected with 125I-IMP and subjected to autoradiographic procedures 20 minutes, and 3 and 24 hours after injection. Differential interference contrast (DIC) images and confocal images were obtained with a confocal laser scanning microscope, and superimposed images were evaluated. Large numbers of silver grains were observed in the interstitium, bronchioles, and alveolar sacs 20 minutes after the injection, and lamellar distribution of the grains was observed on the ciliary surface. The numbers of silver grains in the interstitium and bronchioles had decreased 3 hours after the injection of 125I-IMP, but the numbers of silver grains in the alveolar spaces had not. Although small numbers of silver grains remained in both the bronchioles and alveolar sacs 24 hours after the injection, most of them had washed out. Confocal laser scanning microscopy is considered to be a useful procedure for studying the distribution of radioisotopes by microautoradiography, because it allows clear autoradiographs to be obtained in which tissues and silver grains are perfectly matched and all silver grains are in focus.

A case of local recurrent pheochromocytoma: usefulness of I-123 MIBG early SPECT and maximum intensity projection images.

It has been reported that 10% of patients with pheochromocytoma experience recurrence despite surgery. The authors administered I-123 MIBG to patients with recurrent pheochromocytoma and found that early images are more useful than delayed images to detect recurrence. Early imaging with I-123 MIBG is useful for postoperative follow-up studies in patients with pheochromocytoma and those with possible tumor recurrence.

Synthesis of caged peptides using caged lysine: application to the synthesis of caged AIP, a highly specific inhibitor of calmodulin-dependent protein kinase II.

N(alpha)-Fmoc-N(epsilon)-(2-nitrobenzyloxycarbonyl)-lysine has been prepared and used in the solid-phase synthesis of caged peptides. The synthesized caged AIP (cagedKcagedKALRRQEAVDAL) showed characteristics required for caged peptides including a significantly reduced inhibitory activity to calmodulin-dependent protein kinase II and instantaneous recovery of the activity with photo-irradiation.

Enhancement of antimicrobial activity of neuropeptide Y by N-terminal truncation.

The activity of neuropeptide Y (NPY) against Candida albicans, which was revealed to be fungicidal, was enhanced significantly by the truncation of amino acid residues at the N terminus. The most active peptides (MICs, approximately 1 microM) were about 10-fold more potent than the intact NPY (MIC, approximately 10 microM). The enhancement was weakened by the replacement of the N terminus by negatively charged residues and/or acylation of the alpha-amino group. These results suggest that only the alpha-helical region of NPY is necessary for the antimicrobial activity and that the net charge of the peptide is important for the activity.

Critical amino acid residues of AIP, a highly specific inhibitory peptide of calmodulin-dependent protein kinase II.

The importance of the individual amino acid residues of AIP (KKALRRQEAVDAL), a highly specific inhibitor of calmodulin-dependent protein kinase II (CaMKII), was studied. Replacement of Arg6, Gln7, or Ala9 by other amino acid residues produced a marked increase in the IC50 value. Leu4 and Val10 were also sensitive to replacement, but some hydrophobic amino acids could substitute for these residues. Although replacement of Ala3, Glu8, Ala12, and Leu13 by other residues produced no significant increase in the IC50, the substitution of Lys for Ala3 decreased the IC50. An AIP analog (KKKLRRQEAFDAY), in which Ala3 and Val10 were replaced with Lys and Phe, respectively, showed an IC50 value as low as 4 nM, suggesting that it is a useful tool for studying the physiological roles of CaMKII.

[An evaluation of computed radiography in contrast-enhanced imaging of the gastric area].

The usefulness of computed radiography (CR) in contrast-enhanced imaging of the stomach was evaluated. First, the spatial resolution and density resolution of CR under the conditions of contrast-enhanced imaging of the digestive tract were examined. Spatial resolution was lower, but density resolution was similar, in comparison with those of conventional film screen (FS). Both were better in CR at a low dose. Evaluation of processing conditions for CR using a gastric phantom indicated that the use of N for gamma type (GT) and T for response type (RT) in the left image and the use of A for GT and F for RT in the right image is recommended. Evaluation of the relationship between the stomach segment and response enhancement (RE) showed that RE should not be very strong in cases where segments to be visualized are limited to the lower part of the stomach. With an optimal RE, the image quality of the gastric segment was better in CR than in FS. When dynamic-range control processing was performed in an area overlapped by colon gas, obscure parts became visually recognizable. CR is considered to be useful also for contrast-enhanced imaging of the digestive tract.

Evaluation of uptake and release of technetium-99m MIBI SPECT of pulmonary and mediastinal lesions.

We evaluated the uptake and release of Tc-99m MIBI in 7 benign and 30 malignant pulmonary and mediastinal lesions. Of the 37 patients, 13 underwent surgery; malignant involvement was examined in 21 mediastinal lymph nodes. Tl-201 SPECT was also performed in 10 patients. Tc-99m MIBI SPECT studies were performed on transverse SPECT images acquired 30 minutes and 3 hours after intravenous injection of 600 MBq of Tc-99m MIBI with three gamma camera detectors (GCA-9300A). Regions of interest were set in the area of abnormal uptake of Tc-99m MIBI and in an area of normal tissue in the contralateral lung. The uptake ratio of the lesion in the contralateral normal lung was obtained on the early image (early ratio; ER) as well as the delayed image (delayed ratio; DR). The benign lesions showed significantly lower ER (1.6 +/- 0.3) and DR (1.4 +/- 0.4) than the malignant lesions (1.9 +/- 0.5 and 1.8 +/- 0.5, respectively; both p < 0.05). There was no significant difference in the retention index (RI), calculated as RI = (DR-ER)/ER x 100. The DR obtained with Tl-201 SPECT images was significantly higher than that obtained with Tc-99m MIBI SPECT (p < 0.05). For the detection of mediastinal lymph node metastases, the early images showed sensitivity, specificity, and accuracy of 85.7%, 100%, and 95.2%, respectively, for the delayed images these values were 85.7%, 92.9%, and 90.5%, respectively. These results suggest that the uptake ratio of Tc-99m MIBI is a useful index in assessing benign or malignant pulmonary and mediastinal lesions.

Solid-phase synthesis of caged peptides using tyrosine modified with a photocleavable protecting group: application to the synthesis of caged neuropeptide Y.

A simple method for the synthesis of caged peptides using a novel derivative of tyrosine, N-Fmoc-O-(2-nitrobenzyl)-tyrosine, is described. The derivative of tyrosine can be incorporated at any position in an amino acid sequence by solid-phase peptide synthesis under the condition for Fmoc chemistry, and caged peptides that contain nitrobenzyl group on the side chain of tyrosine residue can be obtained. The nitrobenzyl group can be photocleaved by UV irradiation and the half life of the intermediate during photolysis is approximately 7 microseconds. The method was successfully applied to the synthesis of caged neuropeptide Y (NPY). The binding affinity of the caged NPY for the Y1 receptor was one or two orders of magnitude lower than that of intact NPY, but it increased to the value for intact NPY upon irradiation by UV light.

[Comparative examinations of serum pepsinogen I, II and gastric area using computed radiography in the atrophic gastritis].

The relationship between serum PG I, PG II levels and extent of atrophic gastritis was examined. On 64 patients (male: 32, female: 32, 51.9 years old on average) with established diagnosis of either atrophic gastritis or normal. In the X-ray gastric examination, Fuji Computed Radiography (FCR) was used to obtain clear-cut images of the gastric area. Concerning the serum PG I level, patients in the group with atrophic gastritis showed lower levels than those of the people in the group with no atrophic change, but the variation was wide, and no definite tendency was seen in the relationship between the atrophic change and the serum PG I levels. Concerning the serum PG II level, as the atrophic change progresses, the serum PG II level tended to increase gradually. A significant reduction in the PG I/II ratio was seen in the group with atrophic changes (p < 0.01) in comparison with the group with no atrophic changes, and the PG I/II value tended to decrease. In conclusion, as a relationship between the atrophic change and the serum PG levels had a wide variation, we considered to be difficult by to measure the serum PG level to understand the presence and extent of the atrophic gastritis.

[Assessment of myocardial contraction and relaxation with 99mTc-tetrofosmin multi-gated myocardial SPECT].

Myocardial relaxation at the diastolic phase was not evaluated by multi-gated myocardial SPECT, although myocardial contraction at the systolic phase was studied by percent wall thickening and Bull's eye methods. We make out a myocardial volume curve and report to evaluate the myocardial relaxation using multi-gated myocardial SPECT. The study population consisted of 3 normal human subjects (3 male, 32-37 years old), 10 idiopathic cardiomyopathy, 10 coronary artery disease and 1 hypertensive heart disease combined with aortic regurgitation. All cases were injected 555 MBq of 99mTc-tetrofosmin (Amersham Healthcare Corporation) intravenously at rest. A triple detector gamma-camera (GCA-9300A, Toshiba Medical, Japan) and a data processing computer (GMS-5500A, Toshiba Medical, Japan) were used in this study. A cardiac cycle (R-R interval) was divided by 16 frames (50-80 msec per 1 frame). Eight myocardial volume curves were calculated at the anterior wall, apex and inferior wall of the vertical long axis view and were calculated at the septal wall, apex and lateral wall of the horizontal long axis view, respectively. The patterns of the myocardial volume curves were classified into 5 patterns (Normal pattern (N), Delayed Contraction pattern (DC), Delayed Relaxation pattern (DR), Mixed pattern (M) and Normal pattern with Decreased amplitude (ND)). Myocardial uptake was evaluated visually of grading into severe hypertrophy (5), hypertrophy (4), normal (3), mild hypoperfusion (2), hypoperfusion (1) and perfusion defect (0). We compared patterns of the myocardial volume curves to myocardial uptake in the same segments. It was possible to detect myocardial edge of the total 16 frames with 50-60% threshold in the normal volunteer and in patients with hypertrophic cardiomyopathy and to make a myocardial volume curve. The region of the severe myocardial perfusion defect could be detected with 20% threshold in patients with old myocardial infarction. In comparison with myocardial volume curves and myocardial uptake, 74.6% in the N pattern had a normal uptake (3), 66.7% in the ND pattern had a normal uptake (3), 61.5% in the DC pattern had a hypoperfusion segment (0, 1 or fill-in to normal uptake), 44.4% in the DR pattern had a hypertrophic segment (4, 5 or fill-in to increased uptake). The pattern of myocardial volume curve indicates myocardial contractility and relaxation in each myocardial segment.

[A comparison among 123I-IMP SPECT, EEG and MRI in patients with temporal lobe epilepsy].

N-isopropyl-p-[123I]iodoamphetamine (123I-IMP) single photon emission computed tomography (SPECT), electroencephalography (EEG), and magnetic resonance imaging (MRI) were performed in 19 patients with temporal lobe epilepsy during interictal stage. MRI demonstrated abnormal signal in mesial temporal lobe (hippocampus) in 10 of 19 patients and 123I-IMP SPECT showed a hypoperfusion area in 15 of 19 patients. When compared with EEG and MRI data, disagreement of the affected area was observed in 3 cases. In comparison of EEG and 123I-IMP SPECT data, disagreement of the affected area was observed in 6 cases. Although there were no disagreement in comparison of MRI and 123I-IMP SPECT. We made a reprojection data parallel to the hippocampus in 123I-IMP SPECT. These data demonstrated obviously a hypoperfusion area around the hippocampus. In cases within one month from seizure attack, wide hypoperfusion area was showed on 123I-IMP SPECT in comparison of abnormal signal area on MRI. It could be considered that a reprojection data parallel to the hippocampus was useful to know extent of hypoperfusion area in temporal lobe epilepsy.

Helical scanning CT for hepatocellular carcinoma.

To evaluate the clinical usefulness of helical scanning CT for hepatocellular carcinoma, 39 patients were examined using a Toshiba CT system, the Xforce. Helical scanning CT data were acquired using up to 20 continuous 1.5-sec rotations, with contrast medium bolus injection of 100-150 ml at a rate of 1.5-3 ml/sec, and during a single breath-hold. Scanning was begun 35 to 50 sec after the start of injection. Helical scanning CT permits axial images to be reconstructed at any desired position within the scanned area, and provides images without interslice gaps caused by respiratory movement. Contrast enhanced helical scanning CT permits blood vessels to be clearly displayed following the bolus injection of contrast medium using an automatic injector. Furthermore, the shorter scanning time makes it possible to scan the target area in the desired vascular phase. In all 39 patients who were examined using contrast enhanced helical scanning CT, high-quality three-dimensional images were obtained. Such images were particularly useful for assessing the three-dimensional relationships among high density tumors and vascular structures. In conclusion, helical scanning CT was considered to be useful for hepatocellular carcinoma.

[Clinical evaluation of 99mTc-MAG3 renal imaging: comparison with 99mTc-DTPA, 99mTc-DMSA and 131I-OIH].

The newly developed 99mTc-labeled renal imaging agent, mercaptoacetyltriglycine (MAG3), is a tubular secreted compound. It is expected the most suitable agent to measure renal function and image urinary tract because of the excellent imaging properties and availability of 99mTc and preferred biological qualities of radiohippurates. We examined 15 patients (8 males, 7 females, mean age 55 years old) with renal and urinary tract disease. After drinking 400 ml of water, 185, 370 or 555 MBq of 99mTc-MAG3 was injected with the supine position. Dynamic scans were acquired for 20 minutes on a large field of view gamma camera (ZLC-7500 Siemens Medical Inc.) linked to a computer system (Scintipac-2400 Shimadzu Co.) in a 64 x 64 matrix. Regions of interest (ROI) were placed around each kidney. Curves of 12 seconds frame rate were generated from all two ROIs over the 20 minutes study. The time to peak count (Tmax) and time from peak count to a half count (T1/2) were calculated from the curve data. 99mTc-DTPA renal imaging was examined to the same patients with the same method like 99mTc-MAG3. The renogram of 131I-orthoiodohippurate (OIH) was performed with probe method. The excellent quality of the renal image was obtained with 185-555 MBq of 99mTc-MAG3 compared with the same dose of 99mTc-DTPA respectively. The diagnostic value of 99mTc-MAG3 renal imaging was as well as 99mTc-DMSA renal imaging. The Tmax of 99mTc-MAG3 renogram was same value of that of both 99mTc-DTPA and 131I-OIH renogram. The T1/2 of 99mTc-MAG3 renogram was significantly lower than that of 99mTc-DTPA renogram. The T1/2 of 99mTc-MAG3 renogram was slightly higher that of 131I-OIH renogram. We conclude that the renal imaging of 99mTc-MAG3 was able to acquire the most excellent image and the proper renogram pattern as same as 131I-OIH renogram.

Stabilization of alpha-helix in C-terminal fragments of neuropeptide Y.

To elucidate the alpha-helix-stabilizing effect of amino acids at the helical ends, we prepared analogs of C-terminal fragments of neuropeptide Y (NPY) containing an alpha-helical part. The helix-stabilizing tendency of N-terminal amino acid in NPY (12-36) was found to be as follows: Thr > Ser > Gly > Gln > Cys > Asn > Asp > Val > Phe > Glu > Lys > Tyr > Ala = Trp > His > Arg, suggesting the importance of end capping. The capping effect was not evident when N-termini in NPY (11-36) and NPY (13-36) were replaced. Under the same conditions as those for the receptor binding, [Thr12]NPY (12-36) had about 4-fold higher alpha-helix content than [Arg12]NPY (12-36). However, there was no apparent relationship between the helix content and binding affinity to the Y2 receptor.

[A basic study of multi-gated single photon emission computed tomography with thallium-201].

A Multi-gated SPECT was acquired commonly in 64 x 64 matrix and 8 frames per cardiac cycle (64 x 64/8F). But it was not established that 64 x 64 matrix and 8 frames per cardiac cycle were the most suitable in multi-gated SPECT. Five normal volunteers were examined multi-gated 201Tl SPECT with 5 acquisition modes of 128 x 128 matrix/16 frames, 128 x 128/8F, 64 x 64/32F, 64 x 64/8F using multi-detector SPECT system (GCA-9300). And we calculated percent wall thickening (%WT) [%WT = (ES counts - ED counts)/ED counts] in 9 cases with 64 x 64/8F. The images quality of both 128 x 128/16F and 128 x 128/8F was not clear in compared with images of both 64 x 64/16F and 64 x 64/8F, because the end-diastolic phase of 128 x 128/16F images showed a decreased uptake of 201Tl in the antero-apical region. Although 64 x 64 (8) images had only 8 frames per cardiac cycle, we could observe systolic and diastolic phase and we could calculate %WT. The %WT (M +/- SD) of horizontal long axis images were 48 +/- 15 (sept. basal), 48 +/- 19 (sept. apical), 65 +/- 29 (apex), 49 +/- 22 (lat. apical) and 40 +/- 15 (lat. basal).(ABSTRACT TRUNCATED AT 250 WORDS)

[512 x 2048 matrix whole body scintigraphy with laser imaging system].

Although we could acquire a detailed 512 x 2048 matrix whole body scintigraphy, the 512 x 2048 matrix whole body scintigraphy was divided to the upper and lower half of the body, because a many of CRT system displayed only 1024 x 1024 matrix with non interlace mode. We made 12 dots of normal vertical image distance to 0 dot with laser imaging system (Li-10 Konica medical inc.), and we printed these divided whole body images in the four partition of the film. The lead bar phantom (interval from 6 mm to 3 mm) filled with 99mTcO4- was studied by both 512 x 2048 matrix whole body scanning mode and 256 x 1024 whole body scanning mode in the basic study. And the distance between the lead bar phantom and the gamma camera was changed from 10 mm to 100 mm. We studied 41 patients with metastatic bone tumor (14 breast cancer, 7 lung cancer, 7 prostate cancer, 5 others, 6 unknown origin) clinically. However the 512 x 2048 matrix whole body scan was better quality of images than 256 x 1024 matrix whole body scan at 100 mm distance in the basic study. The abnormal uptake of metastatic sites was shown equally in both 512 x 2048 and 256 x 1024 matrix whole body scintigraphy. The 512 x 2048 matrix whole body scan was better quality of images than 256 x 1024 matrix whole body scan in 26 out of 41 patients, equal in 10 out of 41 patients and worse in 3 out of 41 patients. The matrix size of 512 x 2048 matrix whole body scintigraphy (0.98 mm2) was smaller than that of 256 x 1024 matrix whole body scintigraphy (1.95 mm2).(ABSTRACT TRUNCATED AT 250 WORDS)