Altered postural regulation of foot skin oxygenation and blood flow in patients with type 2 diabetes mellitus.

AIMS: Although skin oxygenation is an important factor in the development and healing of foot ulcers, its regulation was not fully understood. We studied changes in foot skin oxygenation and blood flow during postural changes in patients with type 2 diabetes mellitus. METHODS: Skin oxygenation was measured using transcutaneous oxygen pressure (TcPO(2)) and skin blood flow by laser Doppler flowmetry in 40 patients with type 2 diabetes mellitus without evidence of peripheral arterial disease and 13 healthy control subjects. RESULTS: TcPO(2) in the supine position was significantly lower in patients with type 2 diabetes mellitus compared with control, although skin blood flow was not different. In the sitting position, TcPO(2) significantly increased in control and diabetic patients. The postural change-related increase in TcPO(2) was significantly enhanced in diabetic patients. On the other hand, skin blood blow significantly decreased in the sitting position from the supine position in control subjects but remained stable in diabetic patients. Orthostatic drop in systolic blood pressure correlated negatively with TcPO(2) in the supine position while correlated positively with %change in TcPO(2) and blood flow by postural changes. CONCLUSIONS: The present study demonstrated the dissociated regulation of skin oxygenation and blood flow in response to leg dependency. Impaired postural vasoconstriction was associated with altered regulation of skin oxygenation probably due to sympathetic vascular dysfunction in diabetic patients.

The atypical antipsychotic clozapine impairs insulin secretion by inhibiting glucose metabolism and distal steps in rat pancreatic islets.

AIMS/HYPOTHESIS: Diabetogenic effects of some atypical antipsychotic drugs have been reported, although the mechanisms are not fully understood. We investigated the long-term effects of culturing isolated rat pancreatic islets with atypical antipsychotic clozapine. METHODS: Glucose- and non-glucose-stimulated insulin secretion, glucose metabolism and intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured in islets cultured with or without clozapine. RESULTS: Although acute incubation or 3-day culture with clozapine did not affect glucose-stimulated insulin secretion, clozapine suppressed glucose-stimulated insulin secretion by 53.2% at 1.0 micromol/l (therapeutic concentration) after 7 days of culture. Islet glucose oxidation and [Ca(2+)](i) elevation by high glucose were not affected after 3 days of culture, but clozapine significantly inhibited islet glucose oxidation, ATP production, and [Ca(2+)](i) elevation by high glucose after 7 days of culture. Moreover, 7 days of culture with clozapine inhibited insulin secretion stimulated by: (1) membrane depolarisation induced by high K(+); (2) protein kinase C activation; and (3) mastoparan at 16.7 mmol/l glucose under stringent Ca(2+)-free conditions. Elevation of [Ca(2+)](i) by high K(+)-induced membrane depolarisation was similar in control and clozapine-treated islets. Clozapine, a muscarinic blocker, acutely inhibited carbachol-induced insulin secretion, as did atropine, whereas after 7 days of culture atropine did not have the inhibitory effect shown by clozapine after 7 days. The impairment of glucose-stimulated insulin secretion recovered 3 days after the removal of clozapine treatment. CONCLUSIONS/INTERPRETATION: The present study demonstrated that the atypical antipsychotic drug clozapine directly impaired insulin secretion via multiple sites including glucose metabolism and the distal step in insulin exocytosis in a long-term culture condition. These mechanisms may be involved in the form of diabetes mellitus associated with atypical antipsychotic drugs.

[Treatment and results of interstitial lung diseases in video assisted thoracoscopic lung biopsy].

In order to establish treatment of interstitial lung diseases in video assisted thoracoscopic lung biopsy, we retrospectively reviewed our experiences. The present study included 7 patients with a mean age of 46.4, range from 24 to 61, who were treated at our department from 1996 through 1999. They were 5 men and 2 women. The pathologic diagnosis was nonspecific interstitial pneumonia in 3 patients who responded to steroid therapy. Three other patients had usual interstitial pneumonia. One patient had lymphocytic interstitial pneumonia. No complications occurred. The results indicate that video assisted thoracoscopic lung biopsy is an effective and safe way to diagnose interstitial lung diseases.

Conservative treatment for cervical spondylotic myelopathy. prediction of treatment effects by multivariate analysis.

BACKGROUND CONTEXT: Many studies have suggested only slight effects of conservative treatment on cervical spondylotic myelopathy (CSM), whereas a few reports describe conservative treatment as being effective. This suggested the influence of various factors on treatment outcomes. PURPOSE: We investigated symptomatic changes after conservative treatment in patients based on a clear understanding of the effects and limitations of conservative treatment. STUDY DESIGN: We have encountered cases that showed symptomatic improvement with conservative treatment and became interested in the effectiveness of conservative treatment for CSM and whether other factors affect the results of conservative treatment. PATIENT SAMPLE: We have analyzed the results of conservative treatment for CSM in 69 cases, derived from a population of 101 CSM cases. OUTCOME MEASURES: Symptoms at the time of the first examination were compared with those at the final examination, and the patients were classified into three groups showing improvement, no change or exacerbation. METHODS: Improvement or exacerbation of the symptoms was used as dependent variables and the collected factors as independent variables, and logistic regression was performed on these variables. RESULTS: Multivariate analysis showed significant correlation between clinical outcome and the disease duration and the presence of rigorous conservative treatment. CONCLUSIONS: Conservative treatment for CSM is considered to be effective if it is performed intensively in selected patients. In treating CSM, the therapeutic approach must be selected first in consideration of the patient's disease duration. Conservative treatment must be carried out intensively after sufficient explanation to the patients. Timely surgical intervention is considered to be important if the symptoms show no change or exacerbation with conservative treatment.

Prediction of pulmonary complications after a lobectomy in patients with non-small cell lung cancer.

BACKGROUND: Although the preoperative prediction of pulmonary complications after lung major surgery has been reported in various papers, it still remains unclear. METHODS: Eighty nine patients with stage I-IIIA non-small cell lung cancer (NSCLC) who underwent a complete resection at our institute from 1994-8 were evaluated for the feasibility of making a preoperative prediction of pulmonary complications. All had either a predicted postoperative forced vital capacity (FVC) of >800 ml/m(2) or forced expiratory volume in one second (FEV(1)) of >600 ml/m(2). RESULTS: Postoperative complications occurred in 37 patients (41.2%) but no patients died during the 30 day period after the operation. Pulmonary complications occurred in 20 patients (22.5%). Univariate analysis indicated that the factors significantly related to pulmonary complications were FVC <80%, serum lactate dehydrogenase (LDH) level > or =230 U/l, and arterial oxygen tension (PaO(2)) <10.6 kPa (80 mm Hg). In a multivariate analysis the three independent predictors of pulmonary complications were serum LDH > or =230 U/l (odds ratio (OR) 10.5, 95% CI 1.4 to 77.3), residual volume (RV)/total lung capacity (TLC) > or =30% (OR 6.0, 95% CI 1.1 to 33.7), and PaO(2) <10.6 kPa (OR 5.6, 95% CI 1.4 to 22.2). CONCLUSIONS: The above findings indicate that three factors (serum LDH levels of > or =230 U/l, RV/TLC > or =30%, and PaO(2) <10.6 kPa) may be associated with pulmonary complications in patients undergoing a lobectomy for NSCLC, even though the patient group was relatively small for statistical analysis of such a diverse subject as pulmonary complications.

Pleural retraction and intra-tumoral air-bronchogram as prognostic factors for stage I pulmonary adenocarcinoma following complete resection.

BACKGROUND AND OBJECTIVES: We have retrospectively analyzed the postoperative prognostic factors for 116 patients with stage I adenocarcinoma, with special reference to pleural retraction and intra-tumoral air-bronchogram imaged by computed tomography, which may represent the biological features of pulmonary adenocarcinoma for the retraction of surrounding tissues due to central necrosis and air space-lining growth, respectively. METHODS: The subgroups divided according to the presence of pleural retraction and/or intra-tumoral air-bronchogram on pre-operative CT were compared with respect to the postoperative disease-free survival (DFS) and other clinico-pathological factors. RESULTS: The rates of DFS at 5 years associated with 61 patients with pleural retraction and with 55 patients without pleural retraction were 64.4% and 91.3%, respectively (P = 0.0052), and those associated with 83 patients with air-bronchogram-positive tumors and with 33 patients with air-bronchogram-negative tumors were 81.8% and 64.8%, respectively (P = 0.0040). The DFS at 5 years associated with T1 (73 patients) and T2 (43 patients) were 83.6% and 64.3%, respectively (P = 0.0153). The Cox proportional hazards model analysis revealed that the presence of pleural retraction and the absence of air-bronchogram were independent factors for poor prognosis with relative risks of 7.8 and 5.1, respectively. Pathological T factor was also a significant prognostic factor with a relative risk of 3.2. Seventeen patients with pleural retraction-positive and air-bronchogram-negative tumors showed the high recurrence rate of 47.5% and a poor prognosis with DFS at 5 years of 35.1%. CONCLUSION: These results suggested that, in stage I adenocarcinoma, the degree of malignant potential may be well figured by radiological imaging, with a significant affect on susceptibility of recurrence following complete resection.

Discovery of novel, potent, and selective small-molecule CCR5 antagonists as anti-HIV-1 agents: synthesis and biological evaluation of anilide derivatives with a quaternary ammonium moiety.

The search for new small-molecule CCR5 antagonists by high-throughput screening (HTS) of the Takeda chemical library using [(125)I]RANTES and CHO/CCR5 cells led to the discovery of lead compounds (A, B) with a quaternary ammonium or phosphonium moiety, which were synthesized to investigate new MCP-1 receptor antagonists. A series of novel anilide derivatives 1 with a quaternary ammonium moiety were designed, synthesized, and tested for their CCR5 antagonistic activity. Through the optimization of lead compounds, we have found N,N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6, 7-dihydro-5H-benzocyclohepten-8-yl]carbonyl]amino]benzyl]tetrahydr o-2 H-pyran-4-aminium chloride (1r, TAK-779) as a highly potent and selective nonpeptide CCR5 antagonist with a IC(50) value of 1.4 nM in the binding assay. Compound 1r also inhibited the replication of macrophage (M)-tropic HIV-1 (Ba-L strain) in both MAGI-CCR5 cells and PBMCs with EC(50) values of 1.2 and 3.7 nM, respectively. The synthesis and structure-activity relationships of 1r and its related compounds are detailed.

Evaluations of p53 immunoreactivity, nucleolar organizer regions, and proliferating cell nuclear antigen in non-small cell lung carcinoma.

We examined p53 protein expression, proliferating cell nuclear antigen (PCNA), and argyrophilic nuclear organizer regions (AgNOR), in 102 patients with surgically-treated non-small cell lung cancer (NSCLC). p53 positive cases with DO-1 were defined when more than 10% of the tumor cell nuclei were stained. Mean AgNOR count and PCNA LI were 2.80 and 40.7 and there were no significant differences of AgNOR count and PCNA LI between p53 positive and negative cases. We assessed the relationship between the p53 immunoreactivity and various clinical or pathological parameters. p53 positive rate of stage III disease (46.3%) was significantly higher than that of stage II disease (28.6%). The p53 positive rate of squamous cell carcinoma (42.1%) tended to be higher than that of adenocarcinoma (33.9%). In the survival curves of patients with NSCLC according to the p53 immunoreactivity, there was no significant difference between p53 positive and negative cases. Eight potential prognostic parameters (p53 immunoreactivity, AgNOR count, PCNA LI, sex, age, year of operation, histology, and stage) were also estimated, using univariate and multivariate analysis. In univariate analysis, PCNA LI and AgNOR count, and stage were significantly related to shortened survival. In multivariate analysis, PCNA LI, Age, and stage were independently associated with shortened survival of NSCLC patients. PCNA staining may be more useful than p53 and AgNOR staining in assessing the aggressiveness of surgically-treated NSCLC, although the most useful clinical prognostic parameter should be achieved by the combined analysis of several prognostic indicators.

Prognostic impact of telomerase activity in non-small cell lung cancers.

OBJECTIVE: To evaluate the clinical significance of telomerase activity, particularly in terms of prognostic impact, in non-small cell lung cancer (NSCLC). SUMMARY BACKGROUND DATA: Telomerase activity has been found in various tissues. The activation of telomerase is considered necessary for the immortalization of human tumor cells, including NSCLC. METHODS: The authors studied 103 NSCLC specimens using a polymerase chain reaction based on a telomeric repeat amplification protocol assay. RESULTS: Telomerase activity was detected in 85 (82.5%) of 103 NSCLC specimens but in none of the paired normal lung tissue specimens. More cases of positive telomerase activity were observed in the group with advanced disease and in the group with poorly differentiated tumors. Such factors as the mean age at surgery, sex, smoking, histologic type, and size of tumor extension did not correlate with the telomerase activity. The Kaplan-Meier survival curves in all patients with NSCLC demonstrated that patients with telomerase-positive tumors survived for a significantly shorter period than those with a telomerase-negative tumor (p = 0.0058). According to a multivariate analysis, telomerase activity was identified as an independent prognostic factor (RR = 8.62, p = 0.035). CONCLUSIONS: Telomerase activity was one of the most important prognostic factors in patients with NSCLC, and its potential prognostic implication was independent of tumor stage.

Mediastinal parathyroid cyst: treatment with thoracoscopic surgery--a case report.

Parathyroid cysts are rarely located in the mediastinum. This report describes a 45-year-old man with a mediastinal parathyroid cyst. Video-assisted thoracic surgery was successfully performed to remove the cyst.

Synthesis and biological activity of conformationally restricted analogues of milnacipran: (1S, 2R)-1-phenyl-2-[(R)-1-amino-2-propynyl]-N,N- diethylcyclopropanecarboxamide is a novel class of NMDA receptor channel blocker.

Conformationally restricted analogues of (+/-)-(Z)-2-aminomethyl-1-phenyl-N,N-diethylcyclopropanecarboxamide++ + [milnacipran, (+/-)-1] were designed on the basis of its characteristic cyclopropane structure and were synthesized enantioselectively to develop efficient NMDA receptor antagonists. Among these analogues, (1S,2R)-1-phenyl-2-[(R)-1-amino-2-propynyl]-N, N-diethylcyclopropanecarboxamide (2d) had one of the most potent affinities for the receptor, with a Ki value of 0.29 microM. The blockade of NMDA receptor channels expressed by Xenopus oocytes by 2d was investigated in detail, and 2d was identified as a new class of open channel blocker against this receptor.

Percutaneous suction aspiration and drainage for pyogenic spondylitis.

STUDY DESIGN: Retrospective evaluation of results in 23 cases of early-stage pyogenic spondylitis treated with percutaneous suction aspiration and drainage. OBJECTIVES: To evaluate the efficacy of percutaneous suction aspiration and drainage as a treatment method for early-stage pyogenic spondylitis. SUMMARY OF BACKGROUND DATA: Traditional surgical treatment for pyogenic spondylitis has the disadvantage of increased morbidity caused by the extensive exposure required in the presence of infection. Recently, a few case reports have described minimally invasive treatment for pyogenic spondylitis in which percutaneous suction aspiration was used. However, the efficacy of this new treatment has not yet been evaluated. METHODS: All charts, radiographs, and bacteriologic and histologic findings were reviewed. All 23 patients who received the new treatment were observed clinically and radiographically, to evaluate the efficacy of the treatment. RESULTS: To date, all patients have been observed for more than 2 years. Twenty (87%) of the 23 patients have shown good results according to the evaluation. The causative organism was identified using tissue culturing in 12 (52%) of the 23 patients. The causative organism was Staphylococcus aureus in 8, and Staphylococcus epidermidis, Candida albicans, Pseudomonas aeruginosa, and Propionibacterium acnes in 1 each. Back pain as the major symptom in these patients was relieved within an average of 9.4 days after the operation. However, the patient in whom the spondylitis was caused by Candida albicans has received this new treatment twice without success. CONCLUSIONS: Evaluation of percutaneous suction aspiration with drainage shows that it is an effective treatment for early-stage pyogenic spondylitis.

Vascular endothelial growth factor expression in non-small-cell lung cancer: prognostic significance in squamous cell carcinoma.

BACKGROUND: Recently, some studies have focused on the tumor angiogenesis and its prognostic value. We studied the expression of vascular endothelial growth factor, microvessel counts, and serum concentrations of vascular endothelial growth factor to investigate their association with clinicopathologic factors and prognosis in non-small-cell lung cancer. METHODS: The expression of vascular endothelial growth factor was determined by an immunohistochemical analysis from 91 paraffin specimens of completely resected non-small-cell lung cancers using anti-growth factor polyclonal antibody. Microvessel staining was performed by immunohistochemical analysis with anti-factor VIII-related antigen polyclonal antibody. Measurement of the serum concentrations of vascular endothelial growth factor used the sandwich enzyme-linked immunosorbent assay technique. RESULTS: Expression of vascular endothelial growth factor was detected in 48 of the 91 tumors. The positive ratio was significantly higher in patients with adenocarcinoma than in those with squamous cell carcinoma. The microvessel counts were significantly higher in the patients with nodal metastasis than in those without nodal metastasis. The serum concentrations of vascular endothelial growth factor were also significantly higher in the patients with T3-4 disease than in those with T1-2 disease. The microvessel counts were closely associated with expression of vascular endothelial growth factor. The prognosis of patients with a positive growth factor ratio was significantly worse than that of the patients with a negative ratio (p = 0.002), especially in squamous cell carcinoma. According to a multivariate analysis, only nodal status and expression of vascular endothelial growth factor were found to be independent prognostic factors. CONCLUSIONS: The expression of vascular endothelial growth factor was one of the most important prognostic factors in completely resected non-small-cell lung cancer, especially in squamous cell carcinoma.

Prognostic value of the immunohistochemical detection of p16INK4 expression in nonsmall cell lung carcinoma.

BACKGROUND: The product of the p16INK4/CDKN2/MTS1 (p16) controls the transition from the G1 phase to the S-phase in the cell cycle by inhibiting the phosphorylation of the retinoblastoma gene product. A lack of p16 expression has been reported in various cancer cell lines and tumors; however, there have been only a few reports on the prognostic significance of p16 alteration. The authors studied p16 expression in nonsmall cell lung carcinoma (NSCLC) and also examined its correlation with clinicopathologic features and prognosis. METHODS: p16 expression was determined by immunohistochemical analysis of 115 paraffin specimens of primary NSCLC that were curatively resected. The immunohistochemical study was performed using the labeled streptavidin-biotin method with anti-p16 rabbit polyclonal antibody. RESULTS: Thirty-one of 115 NSCLC specimens (27%) showed negative p16 staining. The frequency of negative p16 expression was significantly higher in squamous cell carcinoma (39.5%) than in adenocarcinoma (20.3%) (P = 0.026). There were no statistically significant differences in the p16 status with respect to age, gender, smoking history, histologic differentiation, or stage of the disease. The Kaplan-Meier survival curves demonstrated that patients with negative p16 expression survived for a significantly shorter period of time than those with positive p16 expression (P = 0.043). p16 status was a significant prognostic factor, especially in patients with early stage disease (Stages I-II) (P = 0.039). CONCLUSIONS: A lack of p16INK4 expression in NSCLC was observed more frequently in squamous cell carcinoma than in adenocarcinoma, and also was found to be closely related to prognosis, especially in patients with early stage squamous cell carcinoma.

Linkage map of Syrian hamster with restriction landmark genomic scanning.

We have constructed the linkage map with precise genetic analysis of the Syrian hamster, Mesocricetus auratus, according to the restriction landmark genomic scanning (RLGS) spot mapping method. Although only 3.2-6.6% of the total RLGS spots between the two strains, ACN and BIO 14.6, showed genetic variance, 572 loci were found to be polymorphic. Out of 569 RLGS loci and 3 other loci, 531 were mapped with the backcross (ACN x BIO 14.6) F1 x BIO 14.6. The cumulative map was 1111.6 cM, indicating that the spots/loci are located throughout the genome at 1.94 cM intervals on average. Thus, RLGS provides us with a rapid tool to construct the genetic map of any species, even if it has less genetic variation.

Construction of high-resolution physical maps from yeast artificial chromosomes using restriction landmark genomic scanning (RLGS).

We have established a new system for chromosome-specific yeast artificial chromosome (YAC) contig construction using restriction landmark genomic scanning (RLGS-based YAC contig mapper). RLGS is a powerful tool for detecting more than 1000 restriction landmarks distributed on an entire genome in one procedure. In this system, RLGS is applied to sorted chromosomes to cover the target chromosome. Using these landmarks as guideposts, chromosome-specific YAC clones are then ordered. In this paper, we report the construction of a map for a human chromosome 21 YAC contig spanning q22.1 using this new approach. Applying RLGS to sorted chromosomes 21 enables detection of approximately 1400 spots (equivalent of 1050 PacI landmarks), covering the entire region of this chromosome. We constructed the 2.5-Mb YAC contig encompassing 21q22.1 with 66 spots (equivalent of 50 PacI landmarks). With this contig map, we could detect two deleted regions and chimerism in the YAC insert DNA. Our results demonstrated the usefulness of this approach for finding DNA alterations of YACs, such as deletions and chimerism.

Permeation of buprenorphine and its 3-alkyl-ester prodrugs through human skin.

PURPOSE: Homologous 3-alkyl-ester prodrugs (C2 to C4) of buprenorphine with decreased crystallinity have been synthesized and evaluated for transdermal delivery commensurate with opioid dependence treatment. METHODS: To assess the influence of derivatization on delivery, the permeation of the prodrugs through human skin was determined in vitro. Prodrug metabolism was measured in human blood and skin supernatant in vitro along with chemical hydrolysis controls. The prodrugs octanol/water partition coefficients were measured. RESULTS: Without exception, the prodrugs were completely hydrolyzed on passing through the skin and appeared as buprenorphine in the receptor compartment. However, using saturation conditions, in no instance did the buprenorphine flux through skin from a prodrug solution exceed the flux of buprenorphine base itself in vitro. Moreover, the flux of the acetyl ester, the least hydrophobic of the prodrugs, was not significantly elevated upon stripping the skin. Whether in blood or the skin supernatant, the prodrugs hydrolyzed in an apparent first-order fashion and rate constants and half-lives were calculated. CONCLUSIONS: We conclude from the results that the prodrugs' very high octanol/water partition coefficients (hydrophobicity) placed them in viable tissue layer controlled diffusion. Consequently, one does not derive the potential flux-increasing benefit of reducing crystallinity that was expected.

Transdermal prodrug concepts: permeation of buprenorphine and its alkyl esters through hairless mouse skin and influence of vehicles.

In vitro skin permeation of buprenorphine (BUP) and three of its alkyl ester prodrugs was evaluated using hairless mouse skin. The three esters selected were the acetyl ester (Ac-BUP), butyl ester (Bu-BUP), and isobutyl ester (Isb-BUP). These drugs were applied on the skin as saturated slurries in three vehicles commonly used to formulate agents for transdermal purposes: propylene glycol, polyethylene glycol 400 (PEG 400), and light mineral oil. Unique solubilities were found for each drug on each vehicle. Fluxes through hairless mouse skin were evaluated for each combination of drug and vehicle using Franz diffusion cells. From PEG 400 formulations, the skin fluxes of BUP, Ac-BUP, Bu-BUP, and Isb-BUP were 0.47 +/- 0.08, 1.64 +/- 0.31, 0.33 +/- 0.05, 0.75 +/- 0.20 micrograms/cm2/h, respectively. Thus, among the three potential prodrugs chosen, only Ac-BUP showed significantly higher skin flux than BUP. There were no inter-vehicle differences in the fluxes from saturated slurries between the vehicles. Moreover, all the esters were detected substantially in the form of regenerated parent drug (BUP) in the receptor compartment. Indeed, only Ac-BUP exited the skin in a measurably intact form, but the fraction escaping metabolism in transit was small (approximately 2%). However, based on drug dispositions in the skin, the regeneration of buprenorphine seems to depend on the alkyl chain length of the ester moiety. The molar percentages of regenerated parent drug in whole drug collected from the skin following the permeation experiments were: Ac-BUP, 9.2%; Bu-BUP, 40.7%; Isb-BUP, 9.6%, respectively. Thus, only Ac-BUP appears promising as a prodrug of buprenorphine, because it is not overly hydrophilic for skin permeation and is also highly metabolized to the parent compound while in the skin.

Antigenic alteration of an anomalous human luteinizing hormone caused by two chorionic gonadotropin-type amino-acid substitutions.

We analyzed the nucleotide sequence of the luteinizing hormone beta subunit (LH beta) in a patient with an anomalous LH. This anomalous LH showed abnormal immunogenicity, but normal bioactivity, suggesting that this variance of antigenicity was caused by amino acid substitution(s). In the anomalous LH, two single amino acid substitutions, Trp(TGG) to Arg(CGG) and Ile(ATC) to Thr(ACC), were found at the codon for the 8th and 15th residue of LH beta. These two substituted amino acid residues of the anomalous LH are identical to those of chorionic gonadotropin, but not to those of LH, although the rest of the region showed the normal sequence of human LH beta. Pedigree analysis by direct DNA sequencing revealed that the parents of the patient and the healthy sister were heterozygotes for the mutation and the patient and the healthy brother were homozygotes.