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Biomedical subjects

A Husain

Publications and source records attributed to A Husain.

At least 37 records · Page 2Linked to original sources
PubMed2000

Angiotensin I-converting enzyme transition state stabilization by HIS1089: evidence for a catalytic mechanism distinct from other gluzincin metalloproteinases.

Angiotensin (Ang) I-converting enzyme (ACE) is a member of the gluzincin family of zinc metalloproteinases that contains two homologous catalytic domains. Both the N- and C-terminal domains are peptidyl-dipeptidases that catalyze Ang II formation and bradykinin degradation. Multiple sequence alignment was used to predict His(1089) as the catalytic residue in human ACE C-domain that, by analogy with the prototypical gluzincin, thermolysin, stabilizes the scissile carbonyl bond through a hydrogen bond during transition state binding. Site-directed mutagenesis was used to change His(1089) to Ala or Leu. At pH 7.5, with Ang I as substrate, k(cat)/K(m) values for these Ala and Leu mutants were 430 and 4,000-fold lower, respectively, compared with wild-type enzyme and were mainly due to a decrease in catalytic rate (k(cat)) with minor effects on ground state substrate binding (K(m)). A 120,000-fold decrease in the binding of lisinopril, a proposed transition state mimic, was also observed with the His(1089) --> Ala mutation. ACE C-domain-dependent cleavage of AcAFAA showed a pH optimum of 8.2. H1089A has a pH optimum of 5.5 with no pH dependence of its catalytic activity in the range 6.5-10.5, indicating that the His(1089) side chain allows ACE to function as an alkaline peptidyl-dipeptidase. Since transition state mutants of other gluzincins show pH optima shifts toward the alkaline, this effect of His(1089) on the ACE pH optimum and its ability to influence transition state binding of the sulfhydryl inhibitor captopril indicate that the catalytic mechanism of ACE is distinct from that of other gluzincins.

Amino Acid Sequence↗
PubMed2000

Affinity chromatography on immobilized "biomimetic" ligands. Synthesis, immobilization and chromatographic assessment of an immunoglobulin G-binding ligand.

A synthetic bifunctional ligand (22/8) comprising a triazine scaffold substituted with 3-aminophenol (22) and 4-amino-1-naphthol (8) has been designed, synthesised, characterised and immobilized on agarose beads to create a robust, highly selective affinity adsorbent for human immunoglobulin G (IgG). Scatchard analysis of the binding isotherm for IgG on immobilized 22/8 (90 micromol 22/8/g moist weight gel) indicated an affinity constant (Ka) of 1.4 x 10(5) M(-1) and a theoretical maximum capacity of 151.9 mg IgG/g moist weight gel. The adsorbent shows similar selectivity to immobilized protein A and binds IgG from a number of species. An apparent capacity of 51.9 mg human IgG/g moist weight gel was observed under the experimental conditions selected for adsorption. Human IgG was eluted with glycine-HCl buffer with a recovery of 67-69% and a purity of 97.3-99.2%, depending on the pH value of the buffer used for elution. Preparative chromatography of IgG from human plasma showed that under the specified conditions, 94.4% of plasma IgG was adsorbed and 60% subsequently eluted with a purity of 92.5%. The immobilized ligand was able to withstand incubation in 1 M NaOH for 7 days without loss of binding capacity for IgG.

Animals↗
PubMed2000

Continent urinary diversion and low-rectal anastomosis in patients undergoing exenterative procedures for recurrent gynecologic malignancies.

OBJECTIVE: The aim of this study was to review the complications associated with continent urinary diversion and associated procedures in patients with gynecologic malignancies. METHODS: We retrospectively reviewed the medical records of all patients who underwent construction of a continent urinary conduit between October 1991 and October 1998 on the Gynecology Service at Memorial Sloan-Kettering Cancer Center. RESULTS: Thirty-three patients were identified, of whom 22 underwent total pelvic exenteration, 8 underwent anterior exenteration, and 3 underwent urinary diversion procedures only. Complications associated with the urinary diversion procedure included ureteral strictures (2), pouch leakage (2), mild hydronephrosis, (6), pyelopnephritis (2), nocturnal incontinence (5), and difficulty with self-catheterization (2). Additional procedures performed concomitantly with continent urinary diversion and exenteration included pelvic reconstruction (18), low-rectal anastomosis (13), and intraoperative radiation therapy (9). The most significant morbidity was seen in patients undergoing concomitant low-rectal anastomosis, in whom the rate of anastomotic leaks was 54% (7 of 13 patients). CONCLUSIONS: Continent urinary diversion can successfully be accomplished at the time of exenteration in patients with recurrent gynecologic malignancies. The rate of major complications related to the urinary diversion is small and most complications can be managed nonsurgically. The greater than 50% rate of anastomotic leaks in patients undergoing concomitant low-rectal anastamosis suggests that such anastomosis should not be undertaken in this group of patients.

Adult↗
PubMed2000

Small cell malignant melanoma: a variant of naevoid melanoma. Clinicopathological features and histological differential diagnosis.

AIMS: To describe the clinical and histopathological features of a rare variant of naevoid melanoma, small cell melanoma, and discuss the histological differential diagnoses. METHODS: The clinical and histological features of cases of malignant melanoma with the histological features of small (non-Merkel like) melanoma were reviewed and documented. In addition, five cases had available material for immunohistochemistry and this was performed using antibodies to the S100 protein and melan-A, and the HMB-45 antibody. RESULTS: There were 15 cases of small cell melanoma from 14 (10 female, four male) patients, aged between 30 and 77 (mean, 48.6) years. The trunk was the most common location. In more than half the cases, the provisional diagnosis was melanoma/borderline lesion. All shared similar histological appearances of an intraepidermal component of in situ melanoma and a dermal component of nests of cells with hyperchromatic nuclei and scanty cytoplasm, usually in tightly packed nests. All components (junctional and intradermal) of the lesions investigated by immunohistochemistry were positive both for S100 protein and melan-A. All junctional components were positive with HMB-45, but with variable staining of the dermal components with this antibody. CONCLUSIONS: Small cell malignant melanoma is postulated to be a distinct histopathological entity and a rare variant of naevoid melanoma. Such lesions can be difficult to interpret and easily missed at scanning magnification because the cells of the dermal component mimic benign naevus cells.

Adult↗
PubMed2000

Analysis of MLH1 and MSH2 expression in ovarian cancer before and after platinum drug-based chemotherapy.

Preclinical studies have demonstrated a relationship between DNA mismatch repair (MMR) status and sensitivity to cisplatin and carboplatin. MMR-deficient cells are resistant to both drugs, and selection for cisplatin resistance in vitro is sometimes accompanied by loss of MMR protein expression. We used immunohistochemical staining techniques to investigate hMLH1 and hMSH2 expression in paired ovarian tumor sections from 54 ovarian cancer patients before and after platinum-based therapy. We sought associations between hMLH1 and hMSH2 protein expression and clinical parameters known to be of prognostic significance as well as response to treatment and overall survival. hMLH1 and hMSH2 staining decreased significantly after platinum-based therapy. The percent of malignant cells that stained positive correlated with the intensity of nuclear staining for both proteins; staining for hMLH1 correlated well with staining for hMSH2. Unexpectedly, expression of nuclear hMLH1 correlated negatively with response to treatment. Expression of nuclear hMLH1 and hMSH2 was positively correlated with pretreatment CA125 level, and expression of nuclear hMSH2 was positively correlated with change in CA125 level after treatment. Tumor stage was associated with expression of nuclear hMSH2, and tumor histological subtype was associated with both hMLH1 and hMSH2 staining. No association was found between expression of either protein and overall survival. These results indicate that the tumor is biologically altered after chemotherapy consistent with treatment-induced selection for cells expressing lower hMLH1 and hMSH2 levels. However, immunohistochemical staining for either hMLH1 or hMSH2 was not highly predictive of drug sensitivity as measured by response or survival.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Regulation of phenylalanine biosynthesis. Studies on the mechanism of phenylalanine binding and feedback inhibition in the Escherichia coli P-protein.

Isothermal titration calorimetry (ITC) and site-directed mutagenesis were used to study the interaction of Phe with (a) the Escherichia coli P-protein, a bifunctional chorismate mutase/prephenate dehydratase that is feedback inhibited by Phe, (b) PDT32, a 32 kDa P-protein fragment (residues 101-386) containing the prephenate dehydratase and regulatory domains, and (c) R12, a C-terminal 12 kDa P-protein fragment (residues 286-386) containing the regulatory domain. DeltaH(total) values for PDT32, which included the heats of Phe binding, conformational change, and dimerization, established that in developing a mechanism for end product feedback inhibition, the P-protein has evolved a ligand recognition domain that exhibits Phe-binding enthalpies comparable to those reported for other full-fledged amino acid receptor proteins. Sequence alignments of R12 with other Phe-binding enzymes identified two highly conserved regions, GALV (residues 309-312) and ESRP (residues 329-332). Site-directed mutagenesis and ITC established that changes in the GALV and ESRP regions affected Phe binding and feedback inhibition to different extents. Mutagenesis further showed that C374 was essential for feedback inhibition, but not for Phe binding, while W338 was involved in Phe binding, but not in the Phe-induced conformational change required for feedback inhibition.

Bacterial Proteins↗
PubMed1999

Role of aromaticity of agonist switches of angiotensin II in the activation of the AT1 receptor.

We have shown previously that the octapeptide angiotensin II (Ang II) activates the AT1 receptor through an induced-fit mechanism (Noda, K., Feng, Y. H., Liu, X. P., Saad, Y., Husain, A., and Karnik, S. S. (1996) Biochemistry 35, 16435-16442). In this activation process, interactions between Tyr4 and Phe8 of Ang II with Asn111 and His256 of the AT1 receptor, respectively, are essential for agonism. Here we show that aromaticity, primarily, and size, secondarily, of the Tyr4 side chain are important in activating the receptor. Activation analysis of AT1 receptor position 111 mutants by various Ang II position 4 analogues suggests that an amino-aromatic bonding interaction operates between the residue Asn111 of the AT1 receptor and Tyr4 of Ang II. Degree and potency of AT1 receptor activation by Ang II can be recreated by a reciprocal exchange of aromatic and amide groups between positions 4 and 111 of Ang II and the AT1 receptor, respectively. In several other bonding combinations, set up between Ang II position 4 analogues and receptor mutants, the gain of affinity is not accompanied by gain of function. Activation analysis of position 256 receptor mutants by Ang II position 8 analogues suggests that aromaticity of Phe8 and His256 side chains is crucial for receptor activation; however, a stacked rather than an amino-aromatic interaction appears to operate at this switch locus. Interaction between these residues, unlike the Tyr4:Asn111 interaction, plays an insignificant role in ligand docking.

Angiotensin I↗
PubMed1999

Phase II trial of intraperitoneal cisplatin and mitoxantrone in patients with persistent ovarian cancer.

OBJECTIVE: The aim of this study was to determine the feasibility and efficacy of intraperitoneal cisplatin and mitoxantrone in patients with very small-volume residual disease at second-look surgery after completion of primary platinum-based intravenous chemotherapy. PATIENTS AND METHODS: Between February 1992 and February 1994, 42 patients were treated with up to five cycles of intraperitoneal cisplatin (100 mg/m2)/mitoxantrone (10 mg/m2). Patients were evaluated for surgically defined response rate and followed for progression-free (PFS) and overall survival (OS) using an intention-to-treat analysis, and grouped according to disease volume at initiation of treatment. RESULTS: The mean age of all patients was 48.5 years. Thirty patients (71%) were Stage III at diagnosis; 18 patients (43%) had microscopic disease at the initiation of IP therapy, and 24 patients (57%) had macroscopic disease. Twenty-eight patients completed three or more cycles of protocol therapy, and 14 patients were changed to standard intravenous therapy after receiving fewer than three cycles of treatment secondary to catheter-related problems (12 patients), cisplatin ototoxicity (1 patient), or withdrawal from study (1 patient). Using an intention-to-treat analysis, the median PFS was 22.5 months, and the median OS of all patients (N = 42) was 47 months (6-72 months) with a median follow-up of 62.7 months. When grouped according to size of disease at initiation of treatment, the OS has not been reached at 62.7 months of follow-up in patients (N = 18) with microscopic disease. CONCLUSIONS: (1) The combination of IP mitoxantrone and cisplatin has an unacceptable catheter failure rate due to mitoxantrone toxicity; (2) PFS and OS is longer in patients with microscopic rather than macroscopic residual disease; and (3) intraperitoneal platinum-based chemotherapy in patients with very small-volume residual disease may result in improved survival.

Adult↗
PubMed1999

Letter

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Journal Article↗
PubMed1999

The inflammatory and cytotoxic effects of a nitric oxide releasing cream on normal skin.

We describe the pro-inflammatory and cytotoxic effects of nitric oxide in vivo in human skin. Nitrite and ascorbic acid were mixed on the skin of 12 normal volunteers, three times daily, to release nitric oxide. Exposure to nitric oxide was varied by randomizing the concentration of nitrite and duration of application. Nitric oxide treated skin showed significant increases in cells expressing CD3, CD4, CD8, CD68, neutrophil elastase, ICAM-1, VCAM-1, nitrosotyrosine, p53, and apoptotic cells compared with skin treated with ascorbic acid alone. There was no significant increase in mast cells. Following application of nitric oxide there were significantly fewer CD1a positive Langerhans cells in the epidermis. These appeared to lose dendritic morphology and migrate from the epidermis. There was no significant difference in staining for Ki-67, a marker related to proliferating cell nuclear antigen, between active and control skin but staining was greater after exposure to higher dose nitric oxide than the low dose. Apoptosis, cytotoxicity, and p53 staining were relatively greater after 48 h exposure than after 24 h. These results suggest that nitric oxide is pro-inflammatory and is toxic to DNA, leading to the accumulation of p53 and subsequent apoptosis. High-dose nitric oxide paradoxically led to a smaller increase in macrophages and T cells than low dose suggesting an immunosuppressive effect of higher levels.

Apoptosis↗
PubMed1999

Dietary intake of pesticides: State of Kuwait total diet study.

The State of Kuwait conducted a total diet study (TDS) to determine the dietary intakes of selected pesticides. This paper reports the results of this study. A national food consumption survey was performed and core food lists for different population groups were established representing the total diet of the Kuwaiti population. Food items (table-ready) were purchased/prepared and analysed for their contents of organophosphate/organochlorine carbamate, benzimidazole and phenylurea pesticides according to the US FDA Pesticide Analytical Manual (PAM I). Dietary intakes of these pesticides are presented for 19 population groups, which range from infants to elderly adults. The intakes of selected population groups are compared with representative findings from the US FDA/TDS and data published from other countries. In general, the average daily intakes were well below acceptable limits, but higher than those reported from developed countries.

Adolescent↗
PubMed1999

Evaluation of the usefulness of testing for p53 mutations in colorectal cancer surveillance for ulcerative colitis.

OBJECTIVE: Immunohistochemical staining for p53 suppressor gene mutations is sensitive and, therefore, has potential for use as a complementary test for dysplasia to improve ulcerative colitis (UC) cancer surveillance program performance. METHODS: A cohort of 95 patients with long standing pan-UC enrolled in a surveillance program was studied. Archival colonic biopsy specimens were stained for p53 mutations and clinical information was obtained from medical records. RESULTS: The 37 patients who developed p53 mutations were significantly more likely to develop dysplasia or cancer (relative risk [RR] 4.53, 95% confidence interval [CI] 2.16-9.48). The p53 mutations developed approximately 8 months before low grade dysplasia, 26 months before high grade dysplasia, and 38 months before cancer. Three of seven cancer patients with p53 mutations had Dukes' stage C or D, whereas only one of five cancer patients without p53 mutations had Dukes' C or D; all three patients who died from metastatic cancer had p53 mutations (three of 37 vs 0 of 58, p < 0.03). Folic acid supplementation had a small, significant protective effect for p53 mutations (RR 0.97, CI 0.94-1.00). CONCLUSION: p53 Mutations 1) are associated with, and likely precede, dysplasia and cancer, 2) are associated with cancer-related mortality, and 3) may possibly be prevented by folic acid supplementation.

Adult↗
PubMed1998

Mechanism of constitutive activation of the AT1 receptor: influence of the size of the agonist switch binding residue Asn(111).

The AT1 receptor is a G-protein-coupled receptor (GPCR); its activation from the basal state (R) requires an interaction between Asn111 in transmembrane helix III (TM-III) of the receptor and the Tyr4 residue of angiotensin II (Ang II). Asn111 to Gly111 mutation (N111G) results in constitutive activation of the AT1 receptor (Noda et al. (1996) Biochemistry, 35, 16435-16442). We show here that replacement of the AT1 receptors TM-III with a topologically identical 16-residue segment (Cys101-Val116) from the AT2 receptor induces constitutive activity, although Asn111 is preserved in the resulting chimera, CR18. Effects of CR18 and N111G mutations are neither additive nor synergistic. The conformation(s) induced in either mutant mimics the partially activated state (R'), and transition to the fully activated R conformation in both no longer requires the Tyr4 of Ang II. Both the R state of the receptor and the Tyr4 Ang II dependence of receptor activation can be reinstated by introduction of a larger sized Phe side chain at the 111 position in CR18, suggesting that the CR18 mutation generated an effect similar to the reduction of side chain size in the N111G mutation. Consistently in the native AT1 receptor, R' conformation is generated by replacement with residues smaller but not larger than the Asn111. However, size substitution of several other TM-III residues in both receptors did not affect transitions between R, R', and R states. Thus, the property responsible for Asn111 function as a conformational switch is neither polarity nor hydrogen bonding potential but the side chain size. We conclude that the fundamental mechanism responsible for constitutive activation of the AT1 receptor is to increase the entropy of the key agonist-switch binding residue, Asn111. As a result, the normally agonist-dependent R --> R' transition occurs spontaneously. This mechanism may be applicable to many other GPCRs.

Amino Acid Sequence↗